Methods and Compositions for the Treatment of Cancer and Related Hyperproliferative Disorders

ABSTRACT

The present invention relates to methods of treating a disease related to cell hyper-proliferation via administration of a therapeutically effective amount of a compound having a general tripartite structure A-B—C. In the tripartite structure A, B, and C are identical or non-identical structures, for example, but not limited to, heterocyclic, phenyl or benzyl ring structures with or without substitutions and are described in detail herein. The methods may utilize particular compounds, for example, having a piperidinyl, a pyrrolinyl or pyridinyl A ring, a thiazole B ring, and a phenyl C ring which may be further substituted independently.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of pending application U.S. Ser. No.14/052,074, filed Oct. 11, 2013, which is a continuation-in-partapplication under 35 U.S.C. §120 of U.S. Ser. No. 14/013,918, filed Aug.29, 2013, now U.S. Pat. No. 9,085,566, which is a continuation-in-partapplication under 35 U.S.C. §120 of application U.S. Ser. No.13/484,702, filed May 31, 2012, now U.S. Pat. No. 8,927,578, which is acontinuation of application U.S. Ser. No. 12/024,530, filed Feb. 1,2008, now U.S. Pat. No. 8,207,196, which is a non-provisionalapplication under 35 U.S.C §119(e) of provisional application U.S. Ser.No. 61/012,310, filed Dec. 7, 2007, now abandoned, and of provisionalapplication U.S. Ser. No. 60/887,994, filed Feb. 2, 2007, now abandoned,the entirety of all of which are hereby incorporated by reference.

FEDERAL FUNDING LEGEND

The present invention utilized federal funding from the NationalInstitutes of Health Grant GM-63115 and Department of Defense Grant No.DAMD17-03-1-0228. The United States Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to the fields of medicine andmolecular biology of metabolic disorders. In particular aspects, thefield of the invention relates to particular compositions for thetreatment of a metabolic disorders, such as obesity or cancer. Incertain aspects, the compositions comprise fatostatin A and its analogsor derivatives.

2. Description of the Related Art

Metabolic syndrome covers many cardiovascular risk factors includinghypertension, dyslipidaemia, obesity, type 2 diabetes, pancreatic β-celldysfunction, and atherosclerosis. A diet varying in fat or carbohydratecontents contributes to energy metabolism of animals including humans.Long chain fatty acids are major source of energy and importantcomponents of the lipids that comprise the cellular membranes. They arederived from food and synthesized de novo from acetyl-CoA. Cholesterolis also derived from food and synthesized from acetyl-CoA. Theconversion of carbohydrates into acylglycerides through de novo fattyacid and cholesterol synthesis involves at least 12 and 23 enzymaticreactions, respectively. Expression levels of the genes encoding theseenzymes are controlled by three transcription factors, designated sterolregulatory element-binding proteins (SREBPs), SREBP-1a, -1c and SREBP-2(Brown and Goldstein, 1997; Osborne, 2000). These membrane-boundproteins are members of a class of the basic helix-loop-helix leucinzipper family of transcription factors (Brown and Goldstein, 1997;Osborne, 2000; Tontonoz et al., 1993). Unlike other leucin zippermembers of transcription factors, SREBPs are synthesized as anER-membrane-bound precursor, which needs to be proteolytically releasedby two proteases bound to the Golgi membrane, Site-1 and Site-2proteases, in order to activate transcription of target genes in thenucleus (Brown and Goldstein, 1997).

The proteolytic activation of SREBPs is tightly regulated by sterolsthrough the interaction with SREBP cleavage-activating protein (SCAP),an ER-membrane-bound escort protein of SREBPs. When sterols accumulatein the ER membranes, the SCAP/SREBP complex fails to exit the ER to theGolgi, and thereby the proteolytic processing of SREBPs is suppressed.SREBPs are key lipogenic transcription factors that govern thehomeostasis of fat metabolism.

The prior art is deficient in the novel compositions and methods usefulfor the treatment of a variety of metabolic disorders. The presentinvention fulfills this longstanding need and desire in the art.

SUMMARY OF THE INVENTION

The present invention is directed to a compound having the generalstructure:

A-B—C

The A ring is a pyridine or a substituted pyridine, a piperidine or asubstituted piperidine, a pyrrolidine or a substituted pyrrolidine, athiazole or a substituted thiazole, a phenyl ring or a substitutedphenyl ring. The B ring is a thioazole or a substituted thioazole, apiperazine or a substituted piperazine, a phenyl ring or a substitutedphenyl ring. The C ring is a phenyl ring or a substituted phenyl ring, apyridine or a substituted pyridine, a thioazole or a substitutedthioazole.

The present invention is directed to a compound having the chemicalstructure

The R₁ substituents are H, halogen, —OH, —O—C₁₋₃ alkoxy, —OC(O)R₃; R₃ isC₁-C₃ alkyl or aryl, —OCH₂—C(O)OR₄; R₄ is H or C₁-C₃ alkyl, —NHR₅; R₅ isH, C₁-C₄ alkyl, alkylcyclopropane, benzyl, —NHC(O)C₁-C₃ amide,—NHC(O)O—R₆ carbamate; R₆ is tert-butyl or benzyl, —NH—SO₂—R₇sulfonamide and R₇ is alkyl or aryl. The R₂ substituents may be alkyl orR₈OC(O)— and R₈ is C₃-C₅ alkyl or aryl.

The present invention directed further to a compound having the chemicalstructure

The R₁ substituents are H, halogen, —OH, —O—C₁₋₃ alkoxy, —OC(O)R₃; R₃ isC₁-C₃ alkyl or aryl, —OCH₂—C(O)OR₄; R₄ is H or C₁-C₃ alkyl, —NHR₅; R₅ isH, C₁-C₄ alkyl, alkylcyclopropane, benzyl, —NHC(O)C₁-C₃ amide,—NHC(O)O—R₆ carbamate; R₆ is tert-butyl or benzyl, —NH—SO₂—R₇sulfonamide and R₇ is alkyl or aryl. The R₂ substituents may be alkyl orR₈OC(O)— and R₈ is C₃-C₅ alkyl or aryl.

The present invention directed further still to a compound having thechemical structure

The R₉ is H, halogen, —OH, —O—C₁-C₃ alkoxy, —OC(O)R₁₁; R₁₁ is C₁-C₃alkyl or aryl, —OCH₂—C(O)OR₁₂; R₁₂ is H or C₁-C₃ alkyl, —NHR₁₃; R₁₃ isH, C₁-C₄ alkyl, alkylcyclopropane, benzyl, —NHC(O)C₁₋₃amide,—NHC(O)O—R₁₄carbamate; R₁₄ is tert-butyl or benzyl, —NH—SO₂—R₁₅sulfonamide; R₁₅ is alkyl or aryl or —SO₂—NH—R₁₆ sulfonamide and R₁₆ isalkyl or aryl. The R₁₀ is nitrogen or methylene. n is 0 or 1 and when nis 1, Z is —C═O. A may have the structure

wherein R₁₇ is H or C₁-C₃ alkyl group.

In a preferred embodiment of the invention, there is a method fortreating a metabolic disorder in an individual, comprising the step ofadministering to the individual a therapeutically effective amount of atleast one compound having the chemical structure:

A-B—C or a pharmaceutically acceptable salt or a stereoisomer thereof ora combination thereof.

In one aspect the metabolic disorder comprises obesity, hyperlipemia,diabetes and complications of diabetes, fatty liver, hypertension,cancer or cardiovascular disease.

In a related embodiment, the individual is provided an additionaltherapy. The additional therapy comprises dietary therapy, physicaltherapy, behavior therapy, surgery, drug therapy, and a combinationthereof. In some aspects, the metabolic disorder is diabetes and theadditional therapy comprises dietary therapy, physical therapy, and drugtherapy.

In another preferred embodiment a compound that isN-(4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenyl)methanesulfonamide,tert-butyl-2-(4-(4-bromophenyl)thiazol-2-yl)pyrrolidine-1-carboxylate,benzyl 2-(4-(4-bromophenyl)thiazol-2-yl)pyrrolidine-1-carboxylate,4-(4-bromophenyl)-2-(pyrrolidin-2-yl)thiazole,4-(4-bromophenyl)-2-(1-propylpyrrolidin-2-yl)thiazole, tert-butyl3-(4-(4-bromophenyl)thiazol-2-yl)piperidine-1-carboxylate, benzyl3-(4-(4-bromophenyl)thiazol-2-yl)piperidine-1-carboxylate,3-(4-(4-bromophenyl)thiazol-2-yl)-1-propyl piperidine, benzyl4-(4-(4-bromophenyl)thiazol-2-yl)piperidine-1-carboxylate,benzyl(R)-2-(4-(4-(methylsulfonamido)phenyl)thiazol-2-yl)pyrrolidine-1-carboxylate,benzyl 3-(4-(4-(methylsulfonamido)phenyl)thiazol-2-yl)piperidine-1-carboxylate, benzyl4-(4-(4-(methylsulfonamido)phenyl)thiazol-2-yl)piperidine-1-carboxylate,4-(3-(pyridin-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)-N-tosylbenzenamine,(4-(5-chloro-2-methylphenyl)piperazin-1-yl)(4-(tosylamino)phenyl)methanone,4-(4-((1-methyl-1H-benzo[d]imidazole-2-yl)methyl)piperazin-1-yl)-N-tozylbenzenamine,3-chloro-4-methyl-N-(6-(4-(3-(trifluoromethyl)benzyl)piperazin-1-yl)pyridin-3-yl)benzenesulfonamide,4-chloro-N-(4-(4-((1-methyl-1H-benzo[d]imidazol-2-yl)methyl)piperazin-1-yl)phenyl)benzenesulfonamide,(Z)-4-(3-cyano-3-(4-(2,4-dimethylphenyl)thiazol-2-yl)allyl)-N-(thiazol-2-yl)benzenesulfonamide,N-(3-(H-imidazo[1,2-a]pyridine-2-yl)phenyl)-4-methyl-2-phenylthiazole-5-carboxamide,N-(3-(benzo[d]thiazol-2-yl)phenyl)isonicotinamide,3-(4-chlorophenyl)-4,5-dihydro-1-phenyl-5-(2-phenylthiazol-4-yl)-1H-pyrazole,N-(4-(6-methylbenzo[d]thiazol-2-yl)phenyl)-2-(N-m-tolylmethylsulfonamido)acetamide,N-(4-(6-methylbenzo[d]thiazol-2-yl)phenyl)-2-(N-p-tolylmethylsulfonamido)acetamide; a pharmaceutically acceptable salt; astereoisomer thereof; and any combination thereof.

In certain embodiment of the invention, there is a method of inhibitinga member of a SREBP pathway in an individual, comprising the step ofproviding to the individual a therapeutically effective amount of atleast one compound of the present invention. In one aspect, the memberof a SREBP pathway is SREBP-1, SREBP-2, or both.

The present invention is also directed to a kit comprising the compounddescribed herein and a container housing the compound.

The present invention is directed further to a method for treating adisease related to cell hyperproliferation in a patient in need thereof,comprising the step of administering to the patient, in apharmaceutically acceptable medium, a therapeutically effective amountof any of the compounds described herein or a pharmaceuticallyacceptable salt; a stereoisomer thereof; and any combination thereof.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter, which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawings.

FIGS. 1A-1B show confirmation of the microarray results by RT-PCR. DU145cells were treated with DMSO (lane 1) and 5 μM fatostatin A (lane 2) for6 hrs (FIG. 1A). Total RNA was then extracted and subjected to RT-PCR.(FIG. 1B) A summary of the RT-PCR and microarray data. ACL, ATP citratelyase; HMG CoAR, 3-hydroxy-3-methyl-glutaryl-CoA reductase; LDLR,low-density lipoprotein receptor, MVD, mevalonate pyrophosphatedecarboxylase; SCD, stearyl-CoA desaturase; INSIG1, insulin-induced gene1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIGS. 2A-2C show that fatostatin A suppresses the ability of endogenousSREBPs to activate a reporter gene. HEK293 cells were co-transfectedwith an SRE-1-driven luciferase reporter (pSRE-Luc) (FIG. 2A) and aβ-gal reporter controlled under an actin promoter (FIG. 2B). Thetransfected cells were treated by varied concentrations of fatostatin Aor DMSO alone in a medium containing lipid-free serum. After 20-hrincubation, luciferase activity was measured, and the data werenormalized by β-galactosidase activity. In FIG. 2C HEK293 cells weretransfected with pCMV-SREBP-1c (1-436) and pSRE-Luc, and the transfectedcells were treated with or without 20 mM fatostatin A in a mediumcontaining lipid-free serum. Each value represents the average of threeindependent experiments.

FIGS. 3A-3H show effects of fatostatin A on SREBP-1 and -2. DU145 cellswere treated with DMSO alone or fatostatin A (1 and 5 μM) for 6 hrs. Thelevels of precursor and mature forms of SREBP-1 (FIG. 3A) or SREBP-2(FIG. 3B) were examined by western blots. Western blots of actin areshown in lower panel as a loading control. (FIGS. 3C-3H) Localization ofSREBP-1 was examined by immunostaining. Cells were treated with DMSOalone (FIGS. 3C-3E) or 5 μM of fatostatin A (FIGS. 3F-3H) and thenstained with DAPI (FIG. 3C and FIG. 3F) or anti-SREBP-1 (FIG. 3D andFIG. 3G).

FIGS. 4A-4G show inhibition of the insulin-induced adipogenesis by siRNAknockdown of the SREBP-1. Two stably transfected clones of 3T3-L1 cellsin which the expression of SREBP-1 were knocked down were establishedand induced to differentiate into adipocytes. The knockdown cells werenot differentiated (FIG. 4D and FIG. 4F), whereas 3T3-L1 cellstransfected with an empty vector (neo) were mostly differentiated intoadipocytes (FIG. 4B). FIGS. 4A, 4C and 4E show the cells without theinsulin induction. FIG. 4G is a Western blot analysis of the clonesindicating the successful knockdown of SREBP-1.

FIGS. 5A-5B demonstrate siRNA knockdown of SREBP-1 blocks theserum-independent growth of DU145 prostate cancer cell. In FIG. 5A twostably transfected clones of DU145 cells in which the expression ofSREBP-1 were knocked down were established and grown in an MEM mediumcontaining no serum, 2% fetal bovine serum (FBS), 2% fat-free fetalbovine serum, or 1 μg/mL of IGF1 for three days. The growth rates weremeasured by WST-1 assays. The knockdown cells failed to grow in the MEMmedium containing no serum, 2% fat-free FBS, or 1 μg/mL of IGF1 butexhibited as much growth as control cells in the presence of serum. Theexperiments were performed in triplicate. FIG. 5B are Western blotsshowing the extents of SREBP-1 knockdown in clones 1 and 2.

FIGS. 6A-6G demonstrate effects of fatostatin A on mice afterfasting/refeeding fat free diet. Mice were injected with 30 mg/kg offatostatin A intraperitoneally daily for the entire experiments startingone day before fasting for 48 hrs followed by feeding fat free diet foranother 48 hrs. Loss of body weight (FIG. 6A) and food intake (FIG. 6B)were determined at the end of the 48-hr feeding. FIG. 6C shows serumconstituents of the treated and control mice. FIG. 6D is arepresentative western blot for SREBP-1 of the liver extracts from 2different mice from the control and fatostatin A treated groups. Theloaded amounts of proteins were normalized. FIG. 6E is a representativewestern blot showing FAS expression (top panel) and a coomassie stainedgel for loading control (bottom) of liver extracts. FIGS. 6F-6Gillustrate activities of FAS and ACC in liver extracts. Data aremeans+SD (n=5); *P<0.05.

FIGS. 7A-7E show effects of two-week treatment of fatostatin A on mice.5-6 month old mice were injected daily for two weeks either with 30mg/kg fatostatin A or 10% DMSO. FIG. 7A shows body weight before andafter the treatment with fatostatin A and FIG. 7B shows the amounts ofweight loss after treatment. FIG. 7C show the serum levels of glucose,cholesterol, and triglycerides (TG) in the treated and control mice.FIG. 7D shows FAS activity in liver extracts. FIG. 7E is arepresentative western blot analysis of the liver extracts of three miceeach for control and treated groups. The loaded amounts of proteins werenormalized. Data are means+SD (n=5); *P<0.05.

FIGS. 8A-8C show effect of fatostatin A on body weight and foodconsumption. Two groups of ob/ob male mice (n=5) were daily injectedintraperitoneally with fatostatin A or 10% DMSO in PBS to control groupsfor four weeks. Mice were fed normal chow and on first day of theexperiment and every day thereafter the weight of the mice and theamount of the food consumed were measured. FIG. 8A is a picture ofrepresentative control and fatostatin A treated mice. FIG. 8B shows theweight of each mouse within each group was measured daily. The averageand variance of the weights are shown. In FIG. 8C food intake wasmeasured every day and was expressed as cumulative food intake per mouseover the 28 days period.

FIGS. 9A-9H demonstrate serum constituents of control and fatostatin Atreated ob/ob mice. Blood was collected from tail veins of overnightfasted mice, and serum was collected after separation from cells.Constituents were determined as described below. The data are shown asmean±SD, n=5 mice in each group.

FIGS. 10A-10D show effect of fatostatin on liver and adipose tissue ofob/ob mice. FIG. 10A shows livers of fatostatin A treated mice (left)and controls (right). FIG. 10B shows histological analyses of frozensections of livers of the control and fatostatin A ob/ob mice stainedwith Oil Red-0 to detect lipid droplets and counter-stained with Mayer'shematoxylin. Livers of three different mice treated with fatostatin A,showing a dramatic decrease in red-stained droplets (top) and controlsshowing an abundance of red-stained lipid droplets compared to thetreated mice (bottom). FIG. 10C shows epididymal fat pads isolated fromfatostatin A treated ob/ob mice (left) and controls (right). FIG. 10Dshows the average weight of livers and epididymal fat pads isolated fromob/ob controls and fatostatin A treated mice. The data are shown asmean±SD, n=5 mice in each group (*P<0.05).

FIGS. 11A-11B shows triglycerides (FIG. 11A) and cholesterol (FIG. 11B)levels in livers of controls and fatostatin A treated ob/ob mice. Lipidswere extracted from livers and triglycerides and cholesterol werequantified as described below. The data are shown as mean±SD, n=5 micein each group (*P=0.0004; †P=0.03).

FIGS. 12A-12D demonstrate that fatostatin A reduces the expressionlevels and activities of lipogenic enzymes. FIG. 12A shows the activityof acetyl-CoA carboxylase (ACC) and FIG. 12B shows fatty acid synthasemeasured in liver extracts of ob/ob mice as described below. FIG. 12C isa Western Blot analysis of liver crude extracts, from three individualob/ob mice, were separated by 4-12% NuPAGE MES gels and probed withdifferent antibodies and detected with ECL. FIG. 12D shows the ratio ofthe intensity of the specific bands, of different lipogenic enzymes fromfatostatin A against control mice after normalization to actin. The dataare shown as mean±SD, n=5 mice in each group (†P=0.005; ‡P=0.002;*P<0.05)

FIG. 13 shows transcript levels of liver lipogenic enzymes in controlrelative to fatostatin A ob/ob mice. mRNA levels for each gene wasnormalized to actin. RNA was isolated from control and fatostatin Atreated mice (n=5) and measured by real-time quantitative RT-PCR.*P<0.05 vs. control.

FIGS. 14A-14F illustrates exemplary compounds 1-66 of the presentinvention.

FIG. 15 demonstrates a standard luciferase reporter gene assay withexemplary analogues 2-18.

FIG. 16 shows a standard luciferase reporter gene assay with exemplaryanalogues 19-34.

FIG. 17 provides a standard luciferase reporter gene assay withexemplary analogues 35-44.

FIGS. 18A-18D show that fatostatin blocks the activation of SREBP.Suppression by fatostatin of the ability of endogenous SREBPs toactivate a luciferase reporter gene in a medium containing lipid-freeserum. CHO-K1 cells were transfected with an SRE-1-driven luciferasereporter (pSRE-Luc) (FIG. 18A). The transfected cells were treated byvaried concentrations of fatostatin in a medium containing lipid-freeserum. Effect of fatostatin on CHO-K1 cells co-transfected withpCMV-SREBP-1c(1-436) and pSRE-Luc in a medium containing lipid-freeserum (FIG. 18B). PLAP-BP2 in transfected CHO-K1 cells remainsmembrane-bound unless it is cleaved by S1P in the Golgi and secretedinto the culture medium (left). Treatment with fatostatin (20 μM) orsterols (10 μg/mL cholesterol and 1 μg/mL 25-hydroxycholesterol) affectcleavage of PLAP-BP2 compared to EtOH controls (FIG. 18C). Western blotanalysis of CHO-K1 cells treated with fatostatin. P and N denote theuncleaved membrane precursor and cleaved nuclear forms of SREBP-2,respectively (FIG. 18D).

FIGS. 19A-19B show that fatostatin blocks the translocation of SREBPsfrom the ER to the Golgi. FIG. 19A is a Western blot analysis showingeffects of brefeldin A on CHO-K1 cells treated with EtOH alone, sterols(10 μg/ml cholesterol and 1 μg/ml 25-hydroxycholesterol), or 20 μMfatostatin. FIG. 19B is a Western blot analysis with anti-SCAP IgG-9D5of cells grown in the absence or presence of 20 μM fatostatin or sterols(10 μg/mL cholesterol and 1 μg/mL 25-hydroxycholesterol). Numbers on theright denote the number of N-linked sugar chains present onprotease-protected SCAP fragments.

FIGS. 20A-20D show the structures of dansyl fatostatin,fatostatin-polyproline linker-biotin conjugate and polyprolinelinker-biotin conjugate and cells treated with the same. FIG. 20Aillustrates how the polyproline linker was inserted for betterprojection of the fatostatin molecule (Sato et al., 2007). In FIG. 20BCHO-K1 cells treated with dansyl fatostatin and ER-tracker red showinglocalization of dansyl fatostatin in the ER. Scale bar=10 μm. FIG. 20Cillustrates the interaction of fatostatin with SCAP, shown by westernblot analysis with anti-SCAP, anti-SREBP-1, anti-SREBP-2, and anti-ATF6antibodies of proteins bound to Neutravidine-agarose beads saturatedwith biotinylated fatostatin in CHO-K1 membrane extract. FIG. 20D showsthat for the competition assay, membrane extracts were preincubated withEtOH alone, cholesterol, or fatostatin.

FIG. 21 shows effects of fatostatin on liver and adipose tissue of ob/obmice. Sections of the livers of fatostatin-treated and control miceshowing red-stained lipid droplets.

FIG. 22 demonstrates a western blot analysis of CHO-K1 cells treatedwith fatostatin. P and N denote the uncleaved membrane precursor andcleaved nuclear forms of SREBP-1, respectively.

FIG. 23 provides an exemplary synthetic scheme of fatostatin, dansylfatostatin and fatostatin-polyproline linker-biotin.

FIG. 24 shows suppression by fatostatin analogues of the ability ofendogenous SREBPs to activate a luciferase reporter gene in a mediumcontaining lipid-free serum. CHO-K1 cells were transfected withpSRE-Luc. The transfected cells were treated by varied concentrations offatostatin, dansyl fatostatin or isopropylamine derivative in a mediumcontaining lipid-free serum.

FIGS. 25A-25D show that fatostatin reduces the expression levels andactivities of lipogenic enzymes. Activity of acetyl-CoA carboxylase(ACC) (FIG. 25A) and fatty acid synthase (FAS) (FIG. 25B) were measuredin liver extracts of ob/ob mice. Western Blot analysis of liver crudeextracts was performed (FIG. 25C). Ratio of the intensity of thespecific bands, of different lipogenic enzymes from fatostatin againstcontrol mice after normalization to actin are shown (FIG. 25D). The dataare shown as mean±SD, n=5 mice in each group (†P=0.005; ‡P=0.002;*P<0.05)

FIG. 26 provides a synthetic scheme of compound 53.

FIG. 27 provides a synthetic scheme of compound 19 and compound 17.

FIG. 28 shows a standard SREBP activation assay with exemplary analogues45-55 and 19.

FIG. 29 shows a standard SREBP activation assay with exemplary analogues56-61 and 19.

FIG. 30 shows a standard SREBP activation assay with exemplary analogues62-66.

FIG. 31 shows an Inhibitory Concentration (sub 50) data of exemplarycompound 53.

FIG. 32 shows an Inhibitory Concentration (sub 50) data of exemplarycompounds 58 and 61.

FIGS. 33A-33B show that compound 19 inhibited the growth of breastcancer cell line SUM 159. Cells were seeded onto 96 well plates at adensity of 10000 cells/well in 100 ul medium containing 2% charcoalstripped serum. After 24 hours, compound 19 was added to the cells atthe indicated concentrations for another 48 hours. Cell viability wasdetermined using WST-1 assay. FIG. 33A: shows the effect of differentconcentrations of compound 19 on cell growth as evident from the changesin the absorbance at A450 nm. FIG. 33B: Expression levels of lipogenicgenes were significantly downregulated by 10 μM treatment of HePG2 cellsas determined by RT PCR analysis (black bar); values are depicted asmeans±SD; *P<0.05

FIGS. 34A-34C show that compound 19 inhibited the growth of human livercancer cell line HePG2. Cells were seeded onto 16 well plates at adensity of 100000 cells/well in medium containing 2% charcoal strippedserum. After 24 hours, compound 19 was added to the cells at theindicated concentrations for another 48 hours. FIG. 34A showsphotographs of control and treated HePG2 cells with 25, 50 and 100 μMcompound 19. FIG. 34B shows a representative Western blot analysisshowing that the treated cells (T) had a decreased level of the matureand active form of SREBP-1 and higher levels of the precursor comparedwith the untreated controls. FIG. 34C shows expression levels oflipogenic genes were significantly downregulated by 10 μM treatment ofHePG2 cells and did not affect INSIG2 which is not known to be regulatedby SREBP, as determined by RT PCR analysis. Values are depicted asmeans±SD; *P<0.05.

FIGS. 35A-35F show that compound 19 inhibited the growth of human acutelymphoblastic leukemia cell line MOLT-4 and human multiple myeloma cellline RPM18226. 10,000 MOLT-4 cells (FIGS. 35A-35C) and 20,000 RPM18226cells (FIGS. 35D-35F) were seeded onto 96 well plate in RPMI 1640 mediumcontaining 5% FBS or fat-free charcoal treated serum (FF-FBS) for 24hours at 37 C. MOLT-4 and RPM18226 cells were treated for additional 48hours with different concentrations of compound 19 (None control withoutDMSO, Vehicle with DMSO, 1, 2, 5, 10 and 20 μM in DMSO; RPM18226 cellsalso were treated with 3 μM in DMSO). At the end of 48 hours cells weresubjected to MTT assay to determine viability. Values are depicted asmeans±SD; *P<0.05.

FIGS. 36A-36F show that compound 17 inhibited the growth of human acutelymphoblastic leukemia cell line MOLT-4 and human multiple myeloma cellline RPM18226. 20,000 MOLT-4 cells (FIGS. 36A-36C) and 20,000 RPM18226cells (FIGS. 36D-36F) cells were seeded onto 96 well plates in RPMI 1640Medium containing 5% Fetal bovine serum (FBS) or 5% fat free charcoaltreated serum (FF-FBS) for 24 hours at 37 C. Cells then were treated foradditional 48 hours with different concentrations of compound 17 (Nonecontrol without DMSO, Vehicle with DMSO, 1, 2, 3, 5, 10 and 20 μM inDMSO). At the end of the 48 hours treatment cells were subjected to MTTassay to determine viability. Values are depicted as means±SD (n=3);**P<0.001; ***P<0.0001.

DETAILED DESCRIPTION OF THE INVENTION

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” may mean at least a second or more. In specificembodiments, aspects of the invention may “consist essentially of” or“consist of” one or more sequences of the invention, for example. Someembodiments of the invention may consist of or consist essentially ofone or more elements, method steps, and/or methods of the invention. Itis contemplated that any method or composition described herein can beimplemented with respect to any other method or composition describedherein.

GENERAL EMBODIMENTS OF THE INVENTION

Metabolic disorders are treated and/or prevented with compounds of thepresent invention. For example, dysregulated biosynthesis of fatty acidsand cholesterols and excessive intake of dietary fat are correlated witha number of medical complications including at least obesity, diabetes,hypertension, and cardiovascular diseases, and in certain aspects theseconditions are treated and/or prevented with a compound of theinvention. Epidemiological evidence indicates that metabolic diseasesincluding obesity also promote the development of an aggressive form ofcancers, including but not limited to prostate cancer.

Upon fat depletion, sterol regulatory element binding proteins (SREBPs)are proteolytically released from the membrane and translocated into thenucleus, where they activate the transcription of the genes involved incholesterol and fatty acid biosynthesis. The present inventionidentifies a synthetic small molecule previously known to block bothadipogenesis and cancer cell growth as a selective inhibitor of theSREBP activation and also provides analogs and derivatives of thatmolecule. The drug-like molecule fatostatin A impairs the proteolyticactivation of SREBPs, thereby reducing the transcription of theirresponsive genes in cells. In mice, fatostatin A blocks the activationof SREBP-1 in the liver, reduces body weight, lowers the levels of bloodcholesterol and glucose, and down-regulates lipogenic enzymes. Fattyacid synthase and acetyl-CoA carboxylase activities and their expressionlevels were decreased in the liver of the treated mice. Fatostatin Aserves as a tool to understand cellular pathways and provides aconsensus molecule as at least starting point for pharmacologicalintervention of metabolic diseases, in certain aspects.

Small molecules that modulate metabolism-related phenotypes serve astools for dissecting the complex associations, in specific embodiments.Fatostatin A causes two distinct phenotypes in cultured mammalian cells:complete inhibition of the insulin-induced adipogenesis of 3T3-L1 mousefibroblast cells; and selective repression of the serum-independentinsulin-like growth factor 1 (IGF1)-dependent growth of DU145 humanprostate cancer cells.

In certain aspects of the invention, fatostatin A selectively blocks theactivation of SREBPs, a key lipogenic transcription factor thatactivates specific genes involved in cholesterol and fatty acidsynthesis. The identification of fatostatin A as an inhibitor of SREBPsis consistent with its anti-adipogenic property, and indicates a role ofSREBPs in the IGF1-dependent growth of prostate cancer.

The present invention concerns fatostatin A as a compound that blocksthe activation of at least SREBP-1, for example as shown in experimentalmice. Administration of fatostatin A into obese ob/ob mice led to weightloss and marked reduction of visceral fat.

The present invention concerns treatment and/or prevention of at leastone symptom of a metabolic disorder. The metabolic disorder may be ofany kind, so long as one of its symptoms is improved or prevented with acompound of the present invention. In particular, though, the metabolicdisease is from one or more inborn errors of metabolism (which may bereferred to as genetic disorders), such as inherited traits that are dueto a defective metabolic enzyme (for example one having one or moremutations or disorders that involve mutations in regulatory proteins andin transport mechanisms).

Generally, metabolic disorders may be defined as disorders that affectenergy production in a cell. Although most metabolic disorders aregenetic, some may be acquired as a result of one or more factors,including diet, toxins, infections, and so forth. Genetic metabolicdisorders may be caused by genetic defects that result in missing orimproperly constructed enzymes necessary for some step in the metabolicprocess of the cell. The largest categories of metabolic disordersinclude the following: 1) glycogen storage diseases (also referred to asglycogenosis or dextrinosis), which include disorders that affectcarbohydrate metabolism; 2) fatty oxidation disorders, which affect fatmetabolism and metabolism of fat components; and 3) mitochondrialdisorders, which affect mitochondria. Examples of glycogen storagediseases (GSD) include at least GSD type I (glucose-6-phosphatasedeficiency; von Gierke's disease); GSD type II (acid maltase deficiency;Pompe's disease); GSD type III (glycogen debrancher deficiency; Cori'sdisease or Forbe's disease); GSD type IV (glycogen branching enzymedeficiency; Andersen disease); GSD type V (muscle glycogen phosphorylasedeficiency; McArdle disease); GSD type VI (liver phosphorylasedeficiency, Hers's disease); GSD type VII (muscle phosphofructokinasedeficiency; Tarui's disease); GSD type IX (phosphorylase kinasedeficiency); and GSD type XI (glucose transporter deficiency;Fanconi-Bickel disease).

Fatty acid metabolism deficiencies may be described as fatty oxidationdisorders or as lipid storage disorders, in certain embodiments. Theymay involve one or more inborn errors of metabolism that result fromenzyme deficiencies that affect the body's ability to oxidize fattyacids for the production of energy within muscles, liver, and other celltypes, for example. Examples of fatty acid metabolism deficienciesinclude at least coenzyme A dehydrogenase deficiencies; other coenzyme Aenzyme deficiencies; carnitine-related disorders; or lipid storagedisorders. Examples of coenzyme A dehydrogenase deficiencies include atleast very long-chain acyl-coenzyme A dehydrogenase deficiency (VLCAD);long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency (LCHAD);medium-chain acyl-coenzyme A dehydrogenase deficiency (MCAD);short-chain acyl-coenzyme A dehydrogenase deficiency (SCAD); and shortchain L-3-hydroxyacyl-coA dehydrogenase deficiency (SCHAD). Examples ofother coenzyme A enzyme deficiencies include at least 2,4 Dienoyl-CoAreductase deficiency; 3-hydroxy-3-methylglutaryl-CoA lyase deficiency;and malonyl-CoA decarboxylase deficiency. Examples of carnitine-relateddeficiencies include at least primary carnitine deficiency;carnitine-acylcarnitine translocase deficiency; carnitinepalmitoyltransferase I deficiency (CPT); and carnitinepalmitoyltransferase II deficiency (CPT). Examples of lipid storagediseases include acid lipase diseases; Wolman disease; cholesteryl esterstorage disease; Gaucher disease; Niemann-Pick disease; Fabry disease;Farber's disease; gangliosidoses; Krabbe disease; and metachromaticleukodystrophy. Other fatty acid metabolism disorders include at leastmitochondrial trifunctional protein deficiency; electron transferflavoprotein (ETF) dehydrogenase deficiency (GAII & MADD); Tangierdisease; and acute fatty liver of pregnancy. Examples of mitochondrialdiseases include at least progressive external ophthalmoplegia (PEO);Diabetes mellitus and deafness (DAD); Leber hereditary optic neuropathy(LHON) Mitochondrial encephalomyopathy, lactic acidosis, and stroke-likesyndrome (MELAS); Myoclonic epilepsy and ragged-red fibers (MERRF);Leigh syndrome; subacute sclerosing encephalopathy; Neuropathy, ataxia,retinitis pigmentosa, and ptosis (NARP); Kearns-Sayre syndrome (KSS);Myoneurogenic gastrointestinal encephalopathy (MNGIE). In particularaspects of the invention, the metabolic disorder is, or has as one ofits complications, one or more of the following: obesity, hyperlipemia,diabetes, fatty liver, hypertension, and cardiovascular disease.

The present invention concerns treatment of a disease related to cellhyperproliferation. In a particular example cell hyperproliferation maybe caused by a cancer or other neoplastic disease or disorder. Withoutbeing limiting, the hyperproliferative disease may be cancer of thebreast, respiratory tract, brain, reproductive organs, prostate,digestive tract, urinary tract, eye, liver, skin, head and neck,thyroid, parathyroid, lymphoma, sarcoma, melanoma, leukemia, multiplemyeloma, or a distant metastasis of a solid tumor.

Exemplary Compositions of the Invention

In a preferred embodiment, there is a method of treatment of metabolicdisorders, the method of treatment comprising the administration of atleast one compound, or pharmaceutically acceptable salts andstereoisomers thereof, having the general formula:

A-B—C

where A, B, and C can be the same or different and each may be a 5-, 6-,or 7-membered ring or a fused bicyclic ring system, the ring being aheterocyclic ring or non-heterocyclic ring, a substituted ring ornon-substituted ring, A, B and C are either directly connected orconnected through an intervening atom chain or linker and said atomchain or linker is a saturated carbon chain or an unsaturated carbonchain with or without additional functional groups.

Preferably, the A ring is a 6-membered heterocyclic ring with oneheteroatom. The A ring may be substituted. In preferred embodiments, thering is a pyridine ring; more preferably, the nitrogen atom of thepyridine ring is in the 4-position or in the 2-position relative to theposition of the B ring. Most preferably, the pyridine ring issubstituted with a n-propyl group on a carbon positioned alpha to thenitrogen heteroatom. In other preferred embodiments, there is a 1-5 atomside chain, more preferably a 1-5 carbon side chain, on the A ring. Inother illustrative and non-limiting embodiments, the A ring may bephenyl, pyrrole, thiophene, furan, pyrimidine, isoquinoline, quinoline,benzofuran, indole, oxazole, naphthyl, piperidine, pyrrolidine,imidazole, imidazol[1,2-a]pyridine, benzoimidazole, thiazole, orbenzothiazole, for example.

In one aspect of the invention, the A ring is a piperidine ring.Preferably, the nitrogen atom of the piperidine ring is in the4-position relative to the position of the B ring. In other relatedaspect, nitrogen atom of the piperidine ring is in the 3-positionrelative to the position of the B ring. The nitrogen atom of thepiperidine may be further substituted and wherein the substitutions areselected from the group consisting of alkyl, sulfoxide, sulfone, alkylor aryl sulfonate, sulfonic acid, and any combination thereof. Forexample, the substitution may be a propyl group, a tert-butyloxycarbonyl(BOC) or a benzyloxycarbonyl group.

In further aspect of the invention, the A ring is a pyrrolidine ring;preferably, the nitrogen atom of the pyrrolidine ring is in the2-position relative to the position of the B ring. The nitrogen atom ofthe pyrrolidine may be further substituted and wherein the substitutionsare selected from the group consisting of alkyl, sulfoxide, sulfone,alkyl or aryl sulfonate, sulfonic acid, and any combination thereof. Forexample, the substitution may be a propyl group, a tert-butyloxycarbonyl(BOC) or a benzyloxycarbonyl group.

Preferably, the B ring is a 5-membered ring with at least twoheteroatoms. The B ring may be substituted. In preferred embodiments,the B ring is a thiazole ring. In other illustrative and non-limitingembodiments, the B ring may be oxazole, imidazol, isooxazole, imidazole,thiphene, furan, pyrimidine, pyrazole, isothiazole, thiazolopyridazine,aryl, or pyrazole, for example. The B ring may further be a 6-memberedring with two heteroatoms. For example, the B ring is a piperazine ring.Preferably, the C ring is a 6-membered ring, most preferably a phenylring. The C ring may be substituted. In preferred embodiments, the Cring is methyl substituted. In other illustrative and non-limitingembodiments, the C ring may be phenyl, pyridine, pyrrole, thiophene,furan, pyrimidine, isoquinoline, quinoline, benzofuran, indole, oxazole,or naphthyl, for example.

Exemplary compounds are provided in FIGS. 14A-14F. Referring to compound1, the n-propyl substituted pyridine ring corresponds to the A ring ofthe general formula, the 2,4-substituted thiazole ring corresponds tothe B ring of the general formula, and the methyl substituted phenylring corresponds to the C ring of the general formula. It should beunderstood that substitutions are permissible at any position in any ofthe A, B, and C rings and any substitutions may be the same or differentfrom any other substitutions. Non-limiting examples of thesubstitutions, in addition to those shown in FIGS. 14A-14F, include thefollowing groups: H (i.e., unsubstituted); hydroxy; C₁₋₁₀ alkyl; C₂₋₁₀alkenyl; C₂₋₁₀ alkynyl; C₃₋₆ cycloalkyl; aryl; heteroaryl; wherein saidalkyl, alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl groups areoptionally substituted with 1-5 groups selected from the groupconsisting of hydroxy, —(C═O)R^(a); —(C═O)OR^(a), —(C═O)H, —(C═O)OH,O(CH₂)_(n)COOR^(a) wherein n=1-10 and wherein R^(a) is a C₁₋₁₀ alkyl,C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, or C₃₋₆ cycloalkyl, aryl, heteroaryl,fluoro, chloro, bromo, iodo, cyano, carboxy, amino, mono-substitutedamino and di-substituted amino, mono-substituted amido anddi-substituted amido and any combination thereof; —(C═O)R^(a);—(C═O)OR^(a), —(C═O)H; —(C═O)OH; —O(CH₂)_(n)COOR^(a) wherein n=1-10 andwherein R^(a) is a C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, or C₃₋₆cycloalkyl, aryl or heteroaryl fluoro, chloro, bromo, iodo; cyano;carboxy; amino; amido, mono- and di-substituted amino having asubstitution selected from the group consisting of C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, C₃₋₆ cycloalkyl, aryl, heteroaryl, sulfoxides,sulfones, sulfonates, alkyl sulfonates, sulfonic acids and anycombination thereof; wherein said alkyl, alkenyl, alkynyl, cycloalkyl,aryl and heteroaryl are optionally substituted with 1-5 groups selectedfrom the group consisting of hydroxy, —(C═O)R^(a), —(C═O)OR^(a),—(C═O)H, —(C═O)OH, —O(CH₂)_(n)COOR^(a) wherein n=1-10 and wherein R^(a)is a C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, or C₃₋₆ cycloalkyl, aryland heteroaryl; fluoro; chloro; bromo; iodo; cyano; carboxy; amino;mono- and di-substituted amino with one or more of C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl groups, and any combination thereof; and, mono-and di-substituted amido having a substitution selected from the groupconsisting of C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₆cycloalkyl, aryl, heteroaryl, sulfoxides, sulfones, sulfonates, alkylsulfonates, sulfonic acids, sulfonates, alkyl sulfonates, sulfonic acidsand any combination thereof; wherein said alkyl, alkenyl, alkynyl,cycloalkyl, aryl and heteroaryl are optionally substituted with 1-5groups selected from the group consisting of hydroxyl; —(C═O)R^(a);—(C═O)OR^(a), —(C═O)H; —(C═O)OH, —O(CH₂)_(n)COOR^(a) wherein n=1-10 andwherein R^(a) is a C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, or C₃₋₆cycloalkyl, aryl or heteroaryl; fluoro; chloro; bromo; iodo; cyano;carboxy; amino; mono- and di-substituted amino with one or more of C₁₋₁₀alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl groups, and any combination thereof.

In the present invention, there is one compound, or pharmaceuticallyacceptable salt or stereoisomer thereof, having the general formula:A-B—C, wherein A, B, and C are the same or different and wherein eachcomprises a 5-, 6-, or 7-membered ring or a fused bicyclic ring system,the ring being a heterocyclic ring or non-heterocyclic ring, asubstituted ring, or non-substituted ring, wherein A, B and C are eitherdirectly connected or connected through an intervening atom chain orlinker and wherein said atom chain or linker is a saturated carbon chainor an unsaturated carbon chain with or without additional functionalgroups, wherein any one, any two, or all three of A, B, and C areunsubstituted or have one or more substitutions, and wherein anysubstitution may be the same or different from any other substitution,and wherein the substitutions are consisting of: a) hydroxy, b) C1-10alkyl, c) C2-10 alkenyl, d) C2-10 alkynyl, e) C3-6 cycloalkyl, f) aryl,g) heteroaryl wherein said substitutions in b), c), d), e), f), and/org) are optionally further substituted with 1-5 groups consisting of: 1)hydroxy, 2) —(C═O)R^(a), 3) —(C═O)OR^(a), 4) —(C═O)H, 5) —(C═O)OH, 6)—O(CH₂)_(n)COOR^(a) wherein n=1-10, 7) halo, 8) cyano, 9) carboxy, 10)amino, 11) mono-substituted amino, 12) di-substituted amino, 13) amido,14) mono-substituted amido; 15) di-substituted amido, and anycombination thereof, wherein in 2), 3), or 6) R^(a) is a C1-10 alkyl,C2-10 alkenyl, C2-10 alkynyl, C3-6 cycloalkyl, aryl, or heteroaryl, h)—(C═O)R^(a), i) —(C═O)OR^(a), j) —(C═O)H, k) —(C═O)OH; l)—O(CH₂)nCOOR^(a) wherein n=1-10, wherein in h), i), or l), R^(a) is aC₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₆ cycloalkyl, aryl orheteroaryl, m) halo, n) cyano, o) carboxy, p) amino, q) mono-substitutedamino, r) di-substituted amino, s) amido, t) mono-substituted amido, andu) di-substituted amido wherein one or more of said mono-substitutedamino, di-substituted amino, mono-substituted amido, and di-substitutedamido have a substitution selected from the group consisting of C1-10alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-6 cycloalkyl, aryl, heteroaryl,sulfoxide, sulfone, sulfonate, alkyl sulfonate, sulfonic acid, and anycombination thereof, wherein in u) said alkyl, alkenyl, alkynyl,cycloalkyl, aryl or heteroaryl are optionally further substituted with1-5 groups selected from the group consisting of: i) hydroxy, ii)—(C═O)R^(a), iii) —(C═O)OR^(a), iv) —(C═O)H, v) —(C═O)OH, vi)—O(CH₂)_(n)COOR^(a) wherein n=1-10, wherein in ii), iii), or vi) R^(a)is a C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₆ cycloalkyl, aryl orheteroaryl, vii) halo, viii) cyano, ix) carboxy, x) amino, xi)mono-substituted amino, xii) di-substituted amino, xiii) amido; xiv)mono-substituted amido, xv) di-substituted amido, and any combinationthereof. Preferably, A is a pyridine or a substituted pyridine, apiperidine or a substituted piperidine, a pyrrolidine or a substitutedpyrrolidine, a thiazole or a substituted thiazole, a phenyl ring or asubstituted phenyl ring; B is a thioazole or a substituted thioazole, apiperazine or a substituted piperazine, a phenyl ring or a substitutedphenyl ring; and C is a phenyl ring or a substituted phenyl ring, apyridine or a substituted pyridine, a thioazole or a substitutedthioazole.

The present invention is directed to a compound having the generalstructure

The R₁ substituents are H, halogen, —OH, —O—C₁₋₃ alkoxy, —OC(O)R₃; R₃ isC₁-C₃ alkyl or aryl, —OCH₂—C(O)OR₄; R₄ is H or C₁-C₃ alkyl, —NHR₅; R₅ isH, C₁-C₄ alkyl, alkylcyclopropane, benzyl, —NHC(O)C₁-C₃ amide,—NHC(O)O—R₆ carbamate; R₆ is tert-butyl or benzyl, —NH—SO₂—R₇sulfonamide and R₇ is alkyl or aryl. The R₂ substituents may be alkyl orR₈OC(O)— and R₈ is C₃-C₅ alkyl or aryl.

The present invention is directed further to a compound having thegeneral structure

The R₁ substituents are H, halogen, —OH, —O—C₁₋₃ alkoxy, —OC(O)R₃; R₃ isC₁-C₃ alkyl or aryl, —OCH₂—C(O)OR₄; R₄ is H or C₁-C₃ alkyl, —NHR₅; R₅ isH, C₁-C₄ alkyl, alkylcyclopropane, benzyl, —NHC(O)C₁-C₃ amide,—NHC(O)O—R₆ carbamate; R₆ is tert-butyl or benzyl, —NH—SO₂—R₇sulfonamide and R₇ is alkyl or aryl. The R₂ substituents may be alkyl orR₈OC(O)— and R₈ is C₃-C₅ alkyl or aryl.

The present invention is directed further still to a compound having thegeneral structure

The R₉ is H, halogen, —OH, —O—C₁-C₃ alkoxy, —OC(O)R₁₁; R₁₁ is C₁-C₃alkyl or aryl, —OCH₂—C(O)OR₁₂; R₁₂ is H or C₁-C₃ alkyl, —NHR₁₃; R₁₃ isH, C₁-C₄ alkyl, alkylcyclopropane, benzyl, —NHC(O)C₁₋₃amide,—NHC(O)O—R₁₄carbamate; R₁₄ is tert-butyl or fluorenylmethyl, —NH—SO₂—R₁₅sulfonamide; R₁₅ is alkyl or aryl or —SO₂—NH—R₁₆ sulfonamide; R₁₆ isalkyl or aryl. The R₁₀ is nitrogen or methylene. n is 0 or 1 and when nis 1, Z is —C═O. A may have the structure

wherein R₁₇ is H or C₁-C₃ alkyl group.

Examples of specific compounds include2-propyl-4-(4-p-tolylthiazol-2-yl)pyridine;4-(4-(4-bromophenyl)thiazol-2-yl)-2-propylpyridine;4-(4-phenylthiazol-2-yl)-2-propylpyridine;4-(4-(4-chlorophenyl)thiazol-2-yl)-2-propylpyridine;4-(4-(4-ethylphenyl)thiazol-2-yl)pyridine;4-(4-p-tolylthiazol-2-yl)pyridine;4-(4-(4-methoxyphenyl)thiazol-2-yl)pyridine;4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenyl benzoate;4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenol; methyl2-(4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenoxy)acetate;2-(4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenoxy)acetic acid;4-(4-chlorophenyl)-2-(3,4-dimethoxyphenyl)thiazole;4-(4-(3,4-dichlorophenyl)thiazol-2-yl)-2-propylpyridine;4-(4-(4-fluorophenyl)thiazol-2-yl)-2-propylpyridine;4-(4-(2,4-difluorophenyl)thiazol-2-yl)-2-propylpyridine;4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)benzenamine;N-isopropyl-4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)benzenamine;N-(4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenyl)acetamide;N-benzyl-4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)benzenamine;N-(cyclopropylmethyl)-4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)benzenamine;4-(4-bromophenyl)-2-(2-propylpyridin-4-yl)thiazole-5-carboxylic acid;methyl 4-(4-bromophenyl)-2-(2-propylpyridin-4-yl)thiazole-5-carboxylate;4-(4-(4-methoxyphenyl)thiazol-2-yl)-2-propylpyridine;4-(4-(3-methoxyphenyl)thiazol-2-yl)-2-propylpyridine;4-(4-(2-methoxyphenyl)thiazol-2-yl)-2-propylpyridine;2-phenyl-4-p-tolylthiazole;3-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenol;2-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenol;4-(4-bromophenyl)-N-isopropyl-2-(2-propylpyridin-4-yl)thiazole-5-carboxamide;4-(4-(4-chlorophenyl)thiazol-2-yl)pyridine;4-(4-(4-chlorophenyl)thiazol-2-yl)-2-ethylpyridine;4-(4-chlorophenyl)-2-phenylthiazole;2-propyl-4-(4-(thiophen-2-yl)thiazol-2-yl)pyridine;4-(4′-methyl[1,1-biphenyl]-4-yl)-2-propyl)pyridine;2-(2-propylpyridin-4-yl)-4-p-tolylthiazole-5-carboxylic acid;2-ethyl-4-(4-p-tolylthiazol-2-yl)pyridine;4-phenyl-2-(2-propylpyridin-4-yl)thiazole-5-carboxylic acid; methyl2-(2-propylpyridin-4-yl)-4-p-tolylthiazole-5-carboxylate; tert-butyl4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenylcarbamate;N-cyclohexyl-4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)benzenamine;4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)-N-tosylbenzenamine;N-(4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenyl)-8-quinolinesulfonamide;N-(4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenyl)-2-thiophenesulfonamide,2-propyl-4-(4-p-tolylthiazol-2-yl)pyridine;4-(4-(4-bromophenyl)thiazol-2-yl)-2-propylpyridine,N-isopropyl-4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)benzenamine, andN-(4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenyl)methanesulfonamide.

Preferred compounds areN-(4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenyl)methanesulfonamide,tert-butyl 2-(4-(4-bromophenyl)thiazol-2-yl)pyrrolidine-1-carboxylate,benzyl 2-(4-(4-bromophenyl)thiazol-2-yl)pyrrolidine-1-carboxylate,4-(4-bromophenyl)-2-(pyrrolidin-2-yl)thiazole,4-(4-bromophenyl)-2-(1-propylpyrrolidin-2-yl)thiazole, tert-butyl3-(4-(4-bromophenyl)thiazol-2-yl)piperidine-1-carboxylate, benzyl3-(4-(4-bromophenyl)thiazol-2-yl)piperidine-1-carboxylate,3-(4-(4-bromophenyl)thiazol-2-yl)-1-propylpiperidine, benzyl4-(4-(4-bromophenyl)thiazol-2-yl)piperidine-1-carboxylate,benzyl(R)-2-(4-(4-(methylsulfonamido)phenyl)thiazol-2-yl)pyrrolidine-1-carboxylate,benzyl3-(4-(4-(methylsulfonamido)phenyl)thiazol-2-yl)piperidine-1-carboxylate,benzyl4-(4-(4-(methylsulfonamido)phenyl)thiazol-2-yl)piperidine-1-carboxylate,4-(3-(pyridin-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)-N-tosylbenzenamine,(4-(5-chloro-2-methylphenyl)piperazin-1-yl)(4-(tosylamino)phenyl)methanone,4-(4-((1-methyl-1H-benzo[d]imidazole-2-yl)methyl)piperazin-1-yl)-N-tozylbenzenamine,3-chloro-4-methyl-N-(6-(4-(3-(trifluoromethyl)benzyl)piperazin-1-yl)pyridin-3-yl)benzenesulfonamide,4-chloro-N-(4-(4-((1-methyl-1H-benzo[d]imidazol-2-yl)methyl)piperazin-1-yl)phenyl)benzenesulfonamide,(Z)-4-(3-cyano-3-(4-(2,4-dimethylphenyl)thiazol-2-yl)allyl)-N-(thiazol-2-yl)benzenesulfonamide,N-(3-(H-imidazo[1,2-a]pyridine-2-yl)phenyl)-4-methyl-2-phenylthiazole-5carboxamide,N-(3-(benzo[d]thiazol-2-yl)phenyl)isonicotinamide,3-(4-chlorophenyl)-4,5-dihydro-1-phenyl-5-(2-phenylthiazol-4-yl)-1H-pyrazole,N-(4-(6-methylbenzo[d]thiazol-2-yl)phenyl)-2-(N-m-tolylmethylsulfonamido)acetamide,N-(4-(6-methylbenzo[d]thiazol-2-yl)phenyl)-2-(N-p-tolylmethylsulfonamido)acetamide;a pharmaceutically acceptable salt; a stereoisomer thereof; and anycombination thereof.

In another preferred embodiment, there is a method of treating a diseaserelated to cell hyper-proliferation in a patient in need thereof. Themethod comprises the step of administering to the patient, in apharmaceutically acceptable medium, a therapeutically effective amountof a compound as disclosed herein or a pharmaceutically acceptable salt;a stereoisomer thereof; and any combination thereof.

The term “alkyl” as used herein refers to a substituting univalent groupderived by conceptual removal of one hydrogen atom from a straight orbranched-chain acyclic saturated hydrocarbon (i.e., —CH₃, —CH₂CH₃,—CH₂CH₂CH₃, —CH(CH₃)₂, —CH₂CH₂CH₂CH₃, —CH₂CH(CH₃)₂, —C(CH₃)₃, etc.).

The term “alkenyl” as used herein refers to a substituting univalentgroup derived by conceptual removal of one hydrogen atom from a straightor branched-chain acyclic unsaturated hydrocarbon containing at leastone carbon-carbon double bond (i.e., —CH═CH₂, —CH═CHCH₃, —C═C(CH₃)₂,—CH₂CH═CH₂, etc.).

The term “alkynyl” as used herein refers to a substituting univalentgroup derived by conceptual removal of one hydrogen atom from a straightor branched-chain acyclic unsaturated hydrocarbon containing at leastone carbon-carbon triple bond (i.e., —C≡CH, —C≡CCH₃, —C≡CCH(CH₃)₂,—CH₂C≡CH, etc.).

The term “aryloxy” as used herein refers to an aryl group with abridging oxygen atom, such as phenoxy (—OC₆H₅), or benzoxy (—OCH₂C₆H₅).“Arylamino means an aryl group with a bridging amine function such as—NHCH₂C₆H₅. “Arylamido” means an aryl group with a bridging amide groupsuch as —(C═O)NHCH₂C₆H₅.

The term “alkylidene” as used herein refers to a substituting bivalentgroup derived from a straight or branched-chain acyclic saturatedhydrocarbon by conceptual removal of two hydrogen atoms from the samecarbon atom (i.e., ═CH₂, ═CHCH₃, ═C(CH₃)₂, etc.).

The term “cycloalkyl” as used herein refers to a substituting univalentgroup derived by conceptual removal of one hydrogen atom from asaturated monocyclic hydrocarbon (i.e., cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, or cycloheptyl).

The term “aryl” as used herein refers to a substituting univalent groupderived by conceptual removal of one hydrogen atom from a monocyclic orbicyclic aromatic hydrocarbon. Examples of aryl groups are phenyl,indenyl, and naphthyl.

The term “heteroaryl” as used herein refers to a substituting univalentgroup derived by the conceptual removal of one hydrogen atom from amonocyclic or bicyclic aromatic ring system containing 1, 2, 3, or 4heteroatoms selected from N, O, or S. Examples of heteroaryl groupsinclude, but are not limited to, pyrrolyl, furyl, thienyl, imidazolyl,pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridyl, pyrimidinyl,pyrazinyl, benzimidazolyl, indolyl, and purinyl. Heteraryl substituentscan be attached at a carbon atom or through the heteroatom. Examples ofmoncyclic heteroaryl groups include pyrrolyl, furyl, thienyl, pyrazolyl,oxazolyl, isoxazolyl, thiazolyl, and pyridyl. Examples of bicyclicheteroaryl groups include pyrimidinyl, pyrazinyl, benzimidazolyl,indolyl, and purinyl. Individual rings may have 5 or 6 atoms. Thus, thisincludes a 4-membered moncyclic heteroaryl group and a 5-memberedmonocylcic heteroaryl group. It also includes a bicyclic heteroarylgroup having one 5-membered ring and one 6-membered ring, and a bicyclicheteroaryl group having two 6-membered rings.

The term “halo” includes iodo, bromo, chloro and fluoro.

The term “substituted” shall be deemed to include multiple degrees ofsubstitution by a substitutent. A substitution occurs where a valence ona chemical group or moiety is satisfied by an atome or functional groupother than hydrogen. In cases of multiple substitutions, the substitutedcompound can be independently substituted by one or more of thedisclosed or claimed substituent moieties, singly or plurally. Byindependently substituted, it is meant that the (two or more)substituents can be the same or different.

The term “pharmaceutically acceptable salt” refers herein to a salt of acompound that possesses the desired pharmacological activity of theparent compound. Such salts include: (1) acid addition salts, formedwith inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, and the like; or formedwith organic acids such as acetic acid, propionic acid, hexanoic acid,cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid,malonic acid, succinic acid, malic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoicacid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonicacid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid,benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-napthalenesulfonicacid, 4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynapthoicacid, salicylic acid, stearic acid, muconic acid, and the like; or (2)salts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like.

The term “stereoisomer” means an isomeric molecule whose atomicconnectivity is the same as one or more other molecules but whose atomicarrangement in space is different. This definition includes enantiomers,diastereomers, cis-isomers, trans-isomers, conformational isomers.

The term “unsubstituted” means all that valences on a chemical group ormoiety are satisfied by hydrogen.

The term “saturated carbon chain” as used herein refers to a straight orbranched-chain acyclic saturated hydrocarbon (i.e., —CH₃, —CH₂—,—CH₂CH₃, —CH₂CH₂—CH₂CH₂CH₃, —CH₂CH₂CH₂, —CH₂CH₂CH₂CH₃, —CH₂CH₂CH₂CH₃—,—CH₂CH(CH₃)₂, —C(CH₃)₃, etc.).

The term “unsaturated carbon chain” as used herein refers to a straightor branched-chain acyclic unsaturated hydrocarbon containing at leastone carbon-carbon double bond (i.e., —CH═CH₂, —CH═CH—, —CH═CHCH₃,—C═C(CH₃)₂, —CH₂CH═CH₂, —CH₂CH═CH— etc.) or at least one carbon-carbontriple bond (i.e., —C≡CH, —C≡C—, —C≡CCH₃, —C≡CCH—, —C≡CCH(CH₃)₂,—CH₂C≡CH, etc.).

The present invention also includes protected derivatives of compoundsdisclosed herein. For example, when compounds of the present inventioncontain groups such as hydroxyl or carbonyl, these groups can beprotected with a suitable protecting group. A list of suitableprotective groups can be found in T. W. Greene, Protective Groups inOrganic Synthesis, John Wiley & Sons, Inc. 1981, the disclosure of whichis incorporated herein by reference in its entirety. The protectedderivatives of compounds of the present invention can be prepared bymethods well known in the art.

The compounds of the present invention may have asymmetric centers,chiral axes, and chiral planes, and occur as racemates, racemicmixtures, and as individual diastereomers, with all possible isomers andmixtures thereof, including optical isomers, being included in thepresent invention. In addition, the compounds disclosed herein may existas tautomers and both tautomeric forms are intended to be encompassed bythe scope of the invention, even though only one tautomeric structuremay be depicted.

In a specific embodiment of the invention, a composition of theinvention targets one or more members of a sterol regulatory elementbinding protein (SREBP) pathway. The pathway relates to the proteolyticrelease of a membrane-bound transcription factor, SREBP, in specificaspects, which facilitates transport from the cytoplasm to the nucleus.There, SREBP binds elements referred to as the sterol regulatoryelements (SREs) present in regulatory regions of the genes that encodeenzymes associated with production of lipids. Upon binding of the SREBPto DNA, transcription of the target gene is modulated, such asupregulated.

Pharmaceutical Preparations

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more compositions of the invention (andadditional agent, where appropriate) dissolved or dispersed in apharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of a pharmaceutical composition thatcontains at least one fatostatin A analog or derivative or additionalactive ingredient will be known to those of skill in the art in light ofthe present disclosure, as exemplified by Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990. Moreover, for animal(e.g., human) administration, it will be understood that preparationsshould meet sterility, pyrogenicity, general safety and purity standardsas required by FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except insofar asany conventional carrier is incompatible with the active ingredient, itsuse in the pharmaceutical compositions is contemplated.

The fatostatin A analog or derivative may comprise different types ofcarriers depending on whether it is to be administered in solid, liquidor aerosol form, and whether it need to be sterile for such routes ofadministration as injection. The present invention can be administeredintravenously, intradermally, transdermally, intrathecally,intraarterially, intraperitoneally, intranasally, intravaginally,intrarectally, topically, intramuscularly, subcutaneously, mucosally,orally, topically, locally, inhalation, e.g., aerosol inhalation,injection, infusion, continuous infusion, localized perfusion bathingtarget cells directly, via a catheter, via a lavage, in cremes, in lipidcompositions, e.g., liposomes, or by other method or any combination ofthe forgoing as would be known to one of ordinary skill in the art (see,for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack PrintingCompany, 1990).

The fatostatin A analog or derivative may be formulated into acomposition in a free base, neutral or salt form. Pharmaceuticallyacceptable salts, include the acid addition salts, e.g., those formedwith the free amino groups of a proteinaceous composition, or which areformed with inorganic acids such as for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric ormandelic acid. Salts formed with the free carboxyl groups can also bederived from inorganic bases such as for example, sodium, potassium,ammonium, calcium or ferric hydroxides; or such organic bases asisopropylamine, trimethylamine, histidine or procaine. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas formulated for parenteral administrations such as injectablesolutions, or aerosols for delivery to the lungs, or formulated foralimentary administrations such as drug release capsules and the like.

Further in accordance with the present invention, the composition of thepresent invention suitable for administration is provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, i.e.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent or carrier is detrimental to the recipient or to thetherapeutic effectiveness of a the composition contained therein, itsuse in administrable composition for use in practicing the methods ofthe present invention is appropriate. Examples of carriers or diluentsinclude fats, oils, water, saline solutions, lipids, liposomes, resins,binders, fillers and the like, or combinations thereof. The compositionmay also comprise various antioxidants to retard oxidation of one ormore component. Additionally, the prevention of the action ofmicroorganisms can be brought about by preservatives such as variousantibacterial and antifungal agents, including but not limited toparabens, e.g., methylparabens, and propylparabens, chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof. In accordancewith the present invention, the composition is combined with the carrierin any convenient and practical manner, i.e., by solution, suspension,emulsification, admixture, encapsulation, absorption and the like. Suchprocedures are routine for those skilled in the art.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach. Examples of stabilizers for use in an thecomposition include buffers, amino acids such as glycine and lysine,carbohydrates such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle compositions that include fatostatin Aanalog or derivative, one or more lipids, and an aqueous solvent. Asused herein, the term “lipid” will be defined to include any of a broadrange of substances that is characteristically insoluble in water andextractable with an organic solvent. This broad class of compounds arewell known to those of skill in the art, and as the term “lipid” is usedherein, it is not limited to any particular structure. Examples includecompounds which contain long-chain aliphatic hydrocarbons and theirderivatives. A lipid may be naturally occurring or synthetic (i.e.,designed or produced by man). However, a lipid is usually a biologicalsubstance. Biological lipids are well known in the art, and include forexample, neutral fats, phospholipids, phosphoglycerides, steroids,terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides,lipids with ether and ester-linked fatty acids and polymerizable lipids,and combinations thereof. Of course, compounds other than thosespecifically described herein that are understood by one of skill in theart as lipids are also encompassed by the compositions and methods ofthe present invention.

One of ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the fatostatin A analog or derivative may bedispersed in a solution containing a lipid, dissolved with a lipid,emulsified with a lipid, mixed with a lipid, combined with a lipid,covalently bonded to a lipid, contained as a suspension in a lipid,contained or complexed with a micelle or liposome, or otherwiseassociated with a lipid or lipid structure by any means known to thoseof ordinary skill in the art. The dispersion may or may not result inthe formation of liposomes.

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. Depending upon the dosage and the route ofadministration, the number of administrations of a preferred dosageand/or an effective amount may vary according to the response of thesubject. The practitioner responsible for administration will, in anyevent, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared is such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

Alimentary Compositions and Formulations

In preferred embodiments of the present invention, the fatostatin Aanalog or derivative is formulated to be administered via an alimentaryroute. Alimentary routes include all possible routes of administrationin which the composition is in direct contact with the alimentary tract.Specifically, the pharmaceutical compositions disclosed herein may beadministered orally, buccally, rectally, or sublingually. As such, thesecompositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

In certain embodiments, the active compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tables,troches, capsules, elixirs, suspensions, syrups, wafers, and the like(Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515;5,580,579; and 5,792,451). The tablets, troches, pills, capsules and thelike may also contain the following: a binder, such as, for example, gumtragacanth, acacia, cornstarch, gelatin or combinations thereof; anexcipient, such as, for example, dicalcium phosphate, mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate or combinations thereof; a disintegrating agent, such as, forexample, corn starch, potato starch, alginic acid or combinationsthereof; a lubricant, such as, for example, magnesium stearate; asweetening agent, such as, for example, sucrose, lactose, saccharin orcombinations thereof; a flavoring agent, such as, for examplepeppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.When the dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar, or both. When the dosage form is a capsule, it maycontain, in addition to materials of the above type, carriers such as aliquid carrier. Gelatin capsules, tablets, or pills may be entericallycoated. Enteric coatings prevent denaturation of the composition in thestomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No.5,629,001. Upon reaching the small intestines, the basic pH thereindissolves the coating and permits the composition to be released andabsorbed by specialized cells, e.g., epithelial enterocytes and Peyer'spatch M cells. A syrup of elixir may contain the active compound sucroseas a sweetening agent methyl and propylparabens as preservatives, a dyeand flavoring, such as cherry or orange flavor. Of course, any materialused in preparing any dosage unit form should be pharmaceutically pureand substantially non-toxic in the amounts employed. In addition, theactive compounds may be incorporated into sustained-release preparationand formulations.

For oral administration the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. For example, a mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

Additional formulations which are suitable for other modes of alimentaryadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum. After insertion, suppositories soften, melt or dissolvein the cavity fluids. In general, for suppositories, traditionalcarriers may include, for example, polyalkylene glycols, triglyceridesor combinations thereof. In certain embodiments, suppositories may beformed from mixtures containing, for example, the active ingredient inthe range of about 0.5% to about 10%, and preferably about 1% to about2%.

Parenteral Compositions and Formulations

In further embodiments, the fatostatin A analog or derivative may beadministered via a parenteral route. As used herein, the term“parenteral” includes routes that bypass the alimentary tract.Specifically, the pharmaceutical compositions disclosed herein may beadministered for example, but not limited to intravenously,intradermally, intramuscularly, intraarterially, intrathecally,subcutaneous, or intraperitoneally U.S. Pat. Nos. 6,537,514, 6,613,308,5,466,468, 5,543,158; 5,641,515; and 5,399,363.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468). In all cases the form must besterile and must be fluid to the extent that easy injectability exists.It must be stable under the conditions of manufacture and storage andmust be preserved against the contaminating action of microorganisms,such as bacteria and fungi. The carrier can be a solvent or dispersionmedium containing, for example, water, ethanol, polyol (i.e., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. A powdered composition is combined with a liquidcarrier such as, e.g., water or a saline solution, with or without astabilizing agent.

In other preferred embodiments of the invention, the active compoundfatostatin A analog or derivative may be formulated for administrationvia various miscellaneous routes, for example, topical (i.e.,transdermal) administration, mucosal administration (intranasal,vaginal, etc.) and/or inhalation.

Pharmaceutical compositions for topical administration may include theactive compound formulated for a medicated application such as anointment, paste, cream or powder. Ointments include all oleaginous,adsorption, emulsion and water-solubly based compositions for topicalapplication, while creams and lotions are those compositions thatinclude an emulsion base only. Topically administered medications maycontain a penetration enhancer to facilitate adsorption of the activeingredients through the skin. Suitable penetration enhancers includeglycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones andluarocapram. Possible bases for compositions for topical applicationinclude polyethylene glycol, lanolin, cold cream and petrolatum as wellas any other suitable absorption, emulsion or water-soluble ointmentbase. Topical preparations may also include emulsifiers, gelling agents,and antimicrobial preservatives as necessary to preserve the activeingredient and provide for a homogenous mixture. Transdermaladministration of the present invention may also comprise the use of a“patch”. For example, the patch may supply one or more active substancesat a predetermined rate and in a continuous manner over a fixed periodof time.

In certain embodiments, the pharmaceutical compositions may be deliveredby eye drops, intranasal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering compositions directly to thelungs via nasal aerosol sprays has been described e.g., in U.S. Pat.Nos. 5,756,353 and 5,804,212. Likewise, the delivery of drugs usingintranasal microparticle resins (Takenaga et al., 1998) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871) are alsowell-known in the pharmaceutical arts. Transmucosal drug delivery in theform of a polytetrafluoroetheylene support matrix is described in U.S.Pat. No. 5,780,045.

The term aerosol refers to a colloidal system of finely divided solid ofliquid particles dispersed in a liquefied or pressurized gas propellant.The typical aerosol of the present invention for inhalation will consistof a suspension of active ingredients in liquid propellant or a mixtureof liquid propellant and a suitable solvent. Suitable propellantsinclude hydrocarbons and hydrocarbon ethers. Suitable containers willvary according to the pressure requirements of the propellant.Administration of the aerosol will vary according to subject's age,weight and the severity and response of the symptoms.

Combination Therapy

In order to increase the effectiveness of a composition of theinvention, an additional therapy may be delivered to an individualhaving a metabolic disorder. For example, an individual that is obesemay be administered a composition of the invention in addition toanother therapy for obesity. Additional obesity therapies includedietary therapy, physical therapy (exercise), drug therapy, surgery, andbehavioral therapy, for example. Exemplary drug therapies include, forexample, Xenical Orlistat®, Phentermine, and Sibutramine (Meridia®).Exemplary surgeries include liposuction and gastric bypass, for example.

For individuals with diabetes, for example, exemplary additionalcompounds for therapy include one or more of the following: Actos(pioglitizone); ACTOSPIus Met; Amaryl (glimepiride); Avandaryl(Avandia+Glimiperide); Avandia (rosiglitazone); Avandamet (rosiglitazonemaleate and metformin hydrochloride); Byettap; Duetact (pioglitazone HCland glimepiride); Galvus (Vildagliptin); Glipizide (Sulfonlyurea);Glucophage (metformin); Glimepiride; Glucovance (glyburide/metformin);Glucotrol XL (glipizide extended release); Glyburide; Glyset (miglitol)glucosidase inhibitor; Januvia (sitagliptin phosphate); Metaglip(glipizide+metformin); Metformin-biguanide; Prandin (repaglinide);Precose (acarbose); Rezulin (troglitazone); Starlix (nateglinide). Othertherapies for diabetes include an improvement in diet and exercise.

Exemplary Measurement of Metabolic Disorder Treatments

In particular aspects of the invention, an individual is given one ormore compositions of the present invention and the individual isassessed for an improvement in at least one symptom of the metabolicdisorder. For example, in particular embodiments when the metabolicdisorder is obesity, an improvement in obesity may be determined duringand/or following treatment with one or more compositions of theinvention. An improvement in obesity may be measured by any standardmeans, but in particular aspects the improvement in obesity is measuredby weight measurement, body mass index (BMI) measurement, and/or bodypart size measurement (such as waist measurement), for example.Exemplary methods for calculating BMI includes dividing a person's bodyweight in kilograms by their height in meters squared (weight [kg]height [m]²). A BMI of 30 or more is considered obese and a BMI between25 to 29.9 is considered overweight. In other aspects of the invention,an individual with diabetes is tested for an improvement followingadministration to the individual of the therapy of the invention. In onespecific embodiment, the monitoring of diabetes occurs by blood test.For example, the blood test may measure the chemical A1C. The higher theblood sugar, the higher the A1C level will be. In some cases,cholesterol (including HDL and/or LDL cholesterol) and/or triglyceridesare measured, such as by standard means in the art. In specific cases, afasting lipoprotein profile is performed, such as by standard means inthe art.

Kits of the Invention

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, the kit comprises a composition suitable fortreatment and/or prevention of one or more metabolic disorders. In otherembodiments of the invention, the kit comprises one or more apparatusesto obtain a sample from an individual. Such an apparatus may be one ormore of a swab, such as a cotton swab, toothpick, scalpel, spatula,syringe, and so forth, for example. In another embodiment, an additionalcompound is provided in the kit, such as an additional compound fortreatment and/or prevention of a metabolic disorder. Any compositionsthat are provided in the kits may be packaged either in aqueous media orin lyophilized form, for example. The container means of the kits willgenerally include at least one vial, test tube, flask, bottle, syringeor other container means, into which a component may be placed, andpreferably, suitably aliquoted. Where there are more than one componentin the kit, the kit also will generally contain a second, third or otheradditional container into which the additional components may beseparately placed.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Fatostatin A Reduces the Expression of SREBP-Responsive Genes

Gene expression profile comparison of the drug-treated and untreatedcells might reveal specific molecular pathways affected by fatostatin A.DU145 cells were treated with fatostatin A or DMSO alone, and extractedmRNA samples were analyzed by Affimetrix DNA microarrays mapping 33,000genes (Table 1).

TABLE 1 Genes known or likely to be controled by SREBPs were regulatedby Fatostatin A showed in Microarray result Genes known to be controlledby SREBP Gene Code Decreased fold Name of genes NM_000527.2 0.574349 lowdensity lipoprotein receptor (LDLR) NM000859.1 0.53-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMG CoA R) NM 0002130.10.353553 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (HMG CoA S)NM_001096.1 0.574349 ATP citrate lyase NM_000664.1 0.574349acetyl-Coenzyme A carboxylase alpha NM_005063.1 0.574349 stearoyl-CoAdesaturase (SCD) NM_002004.1 0.659754 farnesyl pyrophosphate syntlietaseAK000162 0.535887 acetyl-CoA synthetase NM_000431.1 0.5 mevalonatekinase (MVK) NM002461.1 0.329877 mevalonate decarboxylase (MVD) NM003129.2 0.5 squalene epoxidase Genes relevant to sterol/fat synthesisGene Code Decreased fold NM_022977.1 0.707107 fatty-acid-Coenzyme Aligase long chain 4 NM_004457.2 0.707107 fatty-acid-Coenzyme A ligaselong chain 3 NM005931.1 0.659754 fatty acid desaturase 1 NM00193120.659754 dihydrolipoamide S-acetyltransferase AF167438 0.659754 Homosapiens androgen-regulated short- chain dehydrogenaseneductase 1NM006579.1 0.615572 emopamil-bindingprotein (sterol isomerase) D63807.10.615572 lanosterol synthase BC000408.1 0.574349 acetyl-Coenzyme Aacetyltransferase 2 NM_004462.1 0.535887 famesyl-diphosphatefamesyltransferase 1 D85181.1 0.5 sterol-05-desaturase NM_016371.10.466517 hydroxysteroid (17-beta) dehydrogenase 7 NM 005542.1 0.329877Insulin induced gene 1 (INSIG1)

The results showed that 55% of the genes downregulated (<0.7 fold) byfatostatin A are those known or likely to be controlled by sterolregulatory element binding protein (SREBP), including LDL receptor,HMG-CoA reductase, and fatty acid synthase (Horton et al., 2003). Thedownregulation of the representative SREBP-responsive genes wereconfirmed by RT-PCR experiments (FIGS. 1A-1B). These results indicatedthat fatostatin A is a selective inhibitor of the SREBP pathway.

To show that fatostatin A impairs the function of SREBPs, the ability ofendogenous SREBPs to activate transcription of an SREBP-responsivereporter gene was measured in the presence or absence of fatostatin A inHEK293 cells (FIGS. 2A-2B). Fatostatin A decreased in a concentrationdependent manner the activation of the reporter gene in which theexpression of luciferase is controlled by three repeats of sterolregulatory elements. In contrast, fatostatin A failed to impair theability of an exogenously expressed mature form of SREBP-1 (amino acids1-500) to activate the reporter gene activity (FIG. 2C). These resultsindicate that fatostatin A selectively blocks the activation process ofSREBPs in cells.

Example 2 Fatostatin A Blocks the Proteolytic Activation of SREBPS

To examine whether fatostatin A affects the proteolytic activation ofSREBPs, whole cell lysates of DU145 cells treated with fatostatin A wereanalyzed by western blots with an antibody against the NH2 terminus ofSREBP-1 (FIG. 3A). The treatment of fatostatin A decreased the amountsof the 68 KDa mature form of SREBP-1 in a dose-dependent manner, whilethe amounts of the 125 KDa precursor form increased. Similar resultswere obtained for SREBP-2 with an antibody against its COOH terminus(FIG. 3B). These results indicate that fatostatin A directly orindirectly impairs the proteolytic activation of both SREBP isoforms.

The inhibition of the proteolytic activation of SREBPs would impair thenuclear translocation of SREBPs. Effects of fatostatin A on thesubcellular localization of SREBP-1 were analyzed by immunofluoresencemicroscopy with an antibody against the NH2 terminus of SREBP-1. Whencells were treated with DMSO alone, SREBP-1 was localized almostexclusively in the nucleus in a serum-free (fat free) medium (FIGS.3C-3E). In contrast, when the cells were incubated with fatostatin A,the immunofluorescence of SREBP-1 decreased in the nucleus andreciprocally increased outside of the nucleus (FIGS. 3F-3H), indicatingthat fatostatin A inhibits the nuclear localization of SREBP-1.

Example 3 Validation of the Fatostatin Phenotypes by Knocking DownSREBP-1

Fatostatin A causes two phenotypes in cultured cells: (i) inhibition ofthe insulin-induced adipogenesis of 3T3-L1 cells and (ii) repression ofthe serum-independent growth of DU145 prostate cancer cells. The firstphenotype is in complete agreement with the conclusion that fatostatin Ais a blocker of SREBP-1 because of the known role of SREBP-1 inlipogenesis (Tontonoz et al., 1993). To confirm that under thecell-culture condition, the expression of SREBP-1 in 3T3-L1 cells wassilenced by transfecting an expression vector of a small interfering RNA(siRNA) specific for SREBP-1 (FIG. 4G), and the effects of the silencingon the insulin-induced adipogenesis were examined. As expected, theknockdown of the SREBP-1 expression completely blocked the oil dropletformation of 3T3-L1 cells (FIGS. 4D-4F, clones 1 and 2), whereas thecontrol cells transfected with an empty vector (neo; FIG. 4A) showed asmuch fat accumulation as the parental 3T3-L1 cells (FIG. 4B). Theseresults indicate that the fatostatin A-induced phenotype in 3T3-L1 cellsis mediated by the inhibition of SREBP-1.

To test whether the inhibition of SREBP-1 by fatostatin A mediates therepression of serum-independent growth of DU145 cells, the expression ofSREBP-1 in DU145 cells was silenced similarly by transfecting anexpression vector of the SREBP-1-specific siRNA (FIG. 5B). The controlcells transfected with the empty vector (neo) grew in the presence ofeither serum or IGF1, just as the parental DU145 cells did. In contrast,the knockdown cells in which the expression of SREBP-1 is silenced(clones 1 and 2) displayed reduced serum-independent IGF1-driven growthwhereas their serum-dependent growth had little effects (FIG. 5A).

The requirement of SREBP-1 in the serum-independent growth may be due tothe lack of external fat sources in the serum-free medium. Withoutexogenous fatty acids present in the serum, cells need to synthesizefatty acids and cholesterol, the building blocks of membranes, tomaintain the cell growth. To test the importance of fatty acids in thecell growth, the growth of the SREBP-1 knockdown cells was monitored ina fat-free serum medium (FIG. 5A). The SREBP-1 silencing impaired thecell growth in a fat-free medium as much as it did in the serum-freeIGF1-containing medium. These results indicate that fatostatin A blocksthe serum-independent growth of cancer cells through the inhibition ofSREBP-1.

Example 4 Fatostatin A Reduces Body Weight, Lowers Cholesterol andGlucose Levels, and Downregulates Lipogenic Enzymes in Mice

The drug-like chemical structure of fatostatin A prompted the inventorsto investigate its ability to inhibit SREBP-1 in the liver of wholeanimals. The effect of fatostatin A on hepatic SREBP-1 under lipogenicconditions of prolonged fasting (48 hours) followed by feeding fat freehigh carbohydrate diet for another 48 hours was examined. Mice wereintraperitoneally injected with fatostatin A at 30 mg/kg/day for 5 daysstarting one day prior to the 48-hour fasting period. After 48 hours offasting, the treated group lost more weight than the control group did(6.12±0.6 compared to 4.9±0.3 gram/mouse; p=0.01.) (FIG. 6A). Noreduction of food intake or obvious toxicity were observed during thetreatment (FIG. 6B). Interestingly, after 48 hours of refeeding with fatfree high carbohydrate diet, there was a trend of lowering glucoselevels (110±23 compared to 137±14 mg/dl; P=0.06 and cholesterol (93±20compared to 120±19 mg/dl; P=0.12) in the serum of fatostatin A-treatedmice (FIG. 6C). Both HDL and LDL decreased in the treated mice group.However, the decrease in LDL levels appeared to be more significant(16±5 compared to 30±6 mg/dl) (FIG. 6C).

The expression levels of SREBP-1 in the liver extracts were examined bywestern blots. Consistent with the cell culture results, the liverextracts from the mice treated with fatostatin A displayed decreasedamounts of the 68 KDa mature form of SREBP-1 and increased amounts ofthe 125 KDa precursor form (FIG. 6D). The hepatic expression of fattyacid synthase (FAS), a representative of SREBP-1-responsive lipogenicenzymes (Boizard et al., 1998), was also determined after the treatment.Western blot analysis of liver extracts showed that expression levels ofFAS was decreased up to 30% by the fatostatin A treatment (FIG. 6E).Consistent with the reduction of the expression, its enzymatic activityin the extracts was similarly decreased. The activity of acetyl-CoAcarboxylase (ACC) (FIG. 6F), which is also regulated by SREBP-1,decreased in liver extracts as observed for FAS (FIG. 6G). These resultsindicate that fatostatin A blocks the activation of SREBP-1 in mouseliver just as found in the cultured cells.

Longer treatment (two weeks) of another group of mice fed with normaldiet resulted in 10% loss of body weight whereas the control group hadno change of body weight (FIGS. 7A-7B). Food intake was similar betweenboth groups (3.8 and 3.5 g/mouse/day for treated and control mice,respectively). Consistent with the results of mice fed underfasting/refeeding fat free diet, mice fed with normal diet exhibitedsignificantly lower glucose levels and a trend of lower triglyceride(TG) and cholesterol levels in the blood (FIG. 7C). FAS activity and itsprotein level were also decreased about 30% (FIGS. 7D and 7E).

Example 5 Significance of the Present Invention

Bioactive small molecules have proven to be valuable tools for exploringcomplex cellular processes including metabolic pathways. A key regulatorof lipid homeostasis and insulin action is a family of SREBPtranscription factors (Brown and Goldstein, 1997). Small molecules thatmodulate the SREBP functions may find their use in the treatment ofmetabolic diseases and may serve as tools for further molecularunderstanding of the diseases. Our cell-based and animal data suggestthat fatostatin A impairs the expression of lipogenic genes throughdownregulating the amounts of the mature SREBP-1 form in the nucleus. Tothe knowledge of the inventors, fatostatin A represents the firstnon-sterol-like synthetic molecule that inhibits the proteolyticactivation of SREBPs both in cultured cells and mouse liver.

Small molecules that activate SREBP-1 and -2 were been reported(Grand-Perret et al., 2001). These LDL-lowering molecules upregulate theexpression of LDL receptor by stimulating the proteolytic activation ofSREBPs. Although the molecular mechanism of the action has not beenelucidated, data suggested that SCAP is a primary target of themolecules. Unlike these molecules, fatostain A inhibits the activationof SREBPs and downregulates the expression of SREBP-responsive genesincluding the gene of LDL receptor (Table 1).

The animal data of fatostatin A are consistent with the cell cultureresults. In liver extracts of mice treated with fatostatin A underrefeeding fat free diet, there was a significantly lower level of matureSREBP-1 form and a higher level of the precursor form. On the other handand as expected, the level of the mature form was higher than theprecursor form in liver extracts of the control group (Horton et al.,1998). Interestingly, it seems that the there was no change in theoverall amount of the combined forms, and the only difference is at thedistribution between the nuclear (mature) and the cytosolic forms(precursor). These data indicate that fatostatin A may not alter theexpression level of SREBP-1, rather enhances the cleavage process of theprecursor resulting in a decrease in its amount and an increase in themature and active form. The importance of the SREBP-1 cleavage in fatsynthesis has been shown by the experiments using mice deficient inSCAP: the livers of the mice failed to induce the expression of ACC andFAS under refeeding conditions (Liang et al., 2002).

In order to assess the physiological significance of the reduced levelof the nuclear SREBP-1, the levels and activities of ACC and FAS weredetermined. Their activities in liver extracts were downregulated inresponse to fatostatin A treatment. These results are consistent withthe role of SREBP-1 as a regulator of the fatty acid synthesis pathway(Shimano, 2000). Shimano et al. showed that the levels of FAS and ACCwere not induced when SREBP-1^(−/−) mice were fed with high carbohydratediet (Shimano et al., 1997), confirming the role of SREBP-1 inregulating the expression of the lipogenic enzymes.

An interesting observation in the fatostatin A treated mice compared tocontrol is a reduction of body weight and blood glucose. One possibilityfor the reduction in body weight is due to a lower lipogenesis rate, asa result of the downregulation of lipogenic enzymes such as ACC and FAS.In addition, the reduction in malonyl-CoA, the product of ACC and apotent inhibitor of carnitine palmitoyl transferase, may result inenhanced fatty acid oxidation and fat burning. In a specific embodimentof the invention, inhibition of SREBP-1 cleavage by fatostatin Adownregulates lipogenic enzymes, enhances fatty acid oxidation, reducesweight, and increases insulin sensitivity resulting in lowering glucose.

Example 6 Materials

Lipid-depleted serum was prepared as described (Goldstein et al., 1983).Fat-free FBS was obtained from Fisher. Rabbit anti-SREBP-1 (sc-8984) andgoat anti-actin (sc-1616) polyclonal antibody were purchased from SantaCruz Biotechnology. Mouse anti-SREBP-2 polyclonal antibody and mouseanti-FAS antibody were obtained from BD Biosciences. Anti-goat IgG HRPand anti-rabbit IgG HRP were obtained from Promega. ProLong Goldantifade reagent with DAPI was obtained from Molecular Probes InvitrogenDetection Technologies. Anti-rabbit IgG FITC was obtained from ChemiconInternational. Dexamethasone (DEX) and 1-methyl-3-isobutylxanthin (MIX)were obtained from Sigma.

Preparation of Fatostatin A

A mixture of 2-bromo-4′-methylacetophenone (1.22 g, 5.70 mmol) andprothionamide (1.03 g, 5.70 mmol) in ethanol (20 ml) was heated at 70°C. with stirring for 0.5 hour, and then cooled to 0° C. A yellowprecipitate formed was filtered, washed with cold ethanol, and dried togive fatostatin A HBr salt (1.78 g, 83%) as yellow needles: ¹H NMR(DMSO-d₆, 600 MHz) d_(H) 8.88 (d, J=6.2 Hz, 1H), 8.54 (s, 1H), 8.46 (d,J=1.4 Hz, 1H), 8.36 (dd, J=1.4, 6.2 Hz, ¹H), 7.99 (d, J=7.6 Hz, 2H),7.31 (d, J=7.6 Hz, 2H), 3.03 (t, J=7.6 Hz, 2H), 2.35 (s, 3H), 1.80 (m,2H), 0.96 (t, J=7.6, 3H); HRMS (FAB) exact mass calcd for C₁₈H₁₈N₂S+Hrequires m/z 295.1269. found m/z 295.1269.

Cell Culture

DU145 human androgen-independent prostate cancer cells (ATCC) weremaintained in an Eagle's minimum essential medium containing 2 mML-glutamine, 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids,and 1.5 g/L sodium biocarbonate with 10% fetal bovine serum, 100units/mL penicillin, and 100 μg/mL streptomycin sulfate at 37° C. under5% CO₂. 3T3-L1 fibroblasts cells (ATCC) were maintained in a Dulbecco'smodified Eagle's medium containing 5.5 mM glucose, 10% fetal bovineserum, 50 μg/mL gentamycin, 0.5 mM glutamine, and 0.5 μg/mL fungizone at37° C. Human embryonic kidney 293 cells (ATCC) were maintained in aDulbecco's modified Eagle's medium with 10% fetal bovine serum, 100units/mL penicillin, and 100 μg/mL streptomycin sulfate at 37° C. under5% CO₂.

Oligonucleotide Microarray Analysis

DU145 prostate cancer cells were treated with 5 mM of fatostatin A orDMSO alone in the presence of 1 μg/mL of IGF1 for 6 hrs in a serum freemedium, total RNA was extracted in a TRI reagent (Molecular ResearchCenter) and further isolated by RNeasy Mini Kit (Qiagen). Purified mRNAwas analyzed in Baylor College of Medicine Microarray Core Facility byAffymetrix Human Genome U133 Plus 2.0 Array consisting of almost 45,000probe sets representing more than 39,000 transcripts derived fromapproximately 33,000 well-substantiated human genes (Affymetrix, Inc.).

Luciferase Reporter Assay

On day 0, HEK293 cells were plated out in triplicate at a density of5×10³/well onto a 96-well plate in a Dulbecco's modified Eagle's mediumwith 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mLstreptomycin sulfate. On day 2, the cells were transientlyco-transfected with the following plasmids by using Lipofectaminereagent (Invitrogen): 0.4 μg/well pSRE-Luc (an SRE-1-driven luciferasereporter construct; Hua et al., 1995 J Biol Chem 270, 29422-7), and 0.1μg/well a b-gal reporter in which the expression of β-gal is controlledby an actin promoter in a final volume of 150 mL. After incubation for 5hrs at 37° C., the cells were washed with phosphate-buffered saline andthen incubated in 100 μl of Dulbecco's modified Eagle's medium with 10%lipid-depleted serum, 100 units/4 penicillin, and 100 μg/mL streptomycinsulfate in the absence or presence of fatostatin A. After 20 hrs ofincubation, the cells in each well were lysed with 20 μL of 1× ReporterLysis Buffer (Promega), and aliquots were used for measurement ofluciferase (10 μL) and β-galactosidase (10 μL) activities. Forluciferase assay, photon production was detected as counts per second ina Wallac 1420 ARVOsx multilabel counter (PerkinElmer). Forβ-galactosidase assays, hydrolysis of O-nitrophenyl-β-D-galactosidasewas measured after incubation for 0.5 h at 37° C. by a microplate readerat the wave length of 405 nm (Tecan). The luciferase activity (countsper second) was normalized by the activity of β-galactosidase (ODunits). For overexpression of the N-terminal matured form of SREBP-1c,pCMV-SREBP-1c (1-436) was co-transfected with pSRE-Luc. pSRE-Luc andpCMV-SREBP-1c (1-436) were provided by J. L. Goldstein (University ofTexas Southwestern Medical Center).

RT-PCR Experiments

Total RNA was extracted from DU145 cells in TRI reagent (MolecularResearch Center) and isolated with an RNeasy Mini Kit. The RNA samplewas subjected to RT-PCR by using the Access RT-PCR System. RT-PCRreactions contained total RNA, 1 μM of each primer, 0.2 mM dNTP, 1 mMMgSO₄, AMV reverse transcriptase (2 units), and Tfl DNA polymerase (2units) in a final volume of 25 μL. The primer pairs used are as follows:5′-TCA GAC CGG GAC TGC TTG GAC GGC TCA GTC-3′ (SEQ ID NO: 1) and 5′-CCACTT AGG CAG TGG AAC TCG AAG GCC G-3′ (SEQ ID NO: 2) for Low densitylipoprotein receptor (LDLR); 5′-GCC TGC TTG ATA ATA TAT AAA C-3′ (SEQ IDNO: 3) and 5′-CAC TTG AAT TGA GCT TTA G-3′ (SEQ ID NO: 4) forstearoyl-CoA desaturase (SCD); 5′-AAG AAA AAG TGT CAG ACA GCT GG-3′ (SEQID NO: 5) and 5′-TGG ACT GAA GGG GTG TTA GC-3′ (SEQ ID NO: 6) for ATPcitrate lyase (ACL); 5′-GCC CGA CAG TTC TGA ACT GGA ACA-3′ (SEQ ID NO:7) and 5′-GAA CCT GAG ACC TCT CTG AAA GAG-3′ (SEQ ID NO: 8) for3-hydroxy-3-methylglutaryl-CoA reductase (HMG CoA R); 5′-CTG CCT GAC TGCCTC AGC-3′ (SEQ ID NO: 9) and 5′-ACC TCT CCT GAC ACC TGG G-3′ (SEQ IDNO: 10) for mevalonate kinase (MVD); 5′-AAG ACT TCA GGG TAA GTC ATC A-3′(SEQ ID NO: 11) and 5′-CGT GTA TAA TGG TGT CTA TCA G-3′ (SEQ ID NO: 12)for insulin induced gene 1 (INSIG1). The amplification conditions are asfollows: 1 cycle at 94° C. for 4 min, then denatured at 94° C. for 40 s,annealed at 50° C. for 40 s, and extended at 68° C. for 2 min with 22cycles for SCD and HMG CoA R, annealed at 58° C. with 24 cycles for LDLRand INSIG1, or annealed at 60° C. with 24 cycles for ATP citrate lyase(ACL), annealed at 55° C. with 30 cycles for MVD. The amplified DNAswere analyzed by an agarose gel and quantified with the Scion-image(version 4.02) software.

Western Blotting

DU145 prostate cancer cells were seeded on a 6-well plate at a densityof 2×10⁵ cells/well in a serum-free MEM incubated at 37° C. forovernight. The cells were then treated with DMSO or fatostatin A (1 or 5mM) in presence of IGF1 (1 μg/mL). After 6 hrs of incubation, the cellswere harvested in PBS and lysated in an SDS buffer. The samples wereseparated on a 10% SDS-PAGE gel and blotted by using rabbit anti-SREBP-1and anti-SREBP-2 antibodies. The specific bands were visualized by usingenhanced chemiluminescent (ECL) detection reagents (Amersham).

Immunofluorescence Experiments

DU145 prostate cancer cells were seeded on coverslips for overnight in aserum-free MEM, and then treated with 5 mM of fatostatin A or DMSO alonein a serum-free MEM containing IGF1 (1 μg/mL). After 6 hrs ofincubation, the cells were fixed for 20 min in methanol at −20° C. andblocked for 1 hr in a PBS containing 5% milk and 0.1% Tween 20. Thesamples were incubated with rabbit polyclonal anti-SREBP-1 (Santa Cruz:sc-8984) and then fluorescein isothiocyanate-conjugated anti-rabbit IgGantibody (Chemicon Inc). The coverslips were visualized under a NikonTE200 fluorescence microscope at ×400 magnification with appropriatefilters for fluorescence detection.

siRNA knockdown of SREBPs

Complimentary oligonucleotides derived from the sequence of the SREBP-1gene (512-531), 5′-GAT CCC CGC CAC ATT GAG CTC CTC TCT TCA AGA GAG AGAGGA GCT CAA TGT GGC TTT TTG GAAA-3′ (SEQ ID NO: 13), and 5′-AGC TTT TCCAAA AAG CCA CAT TGA GCT CCT CTC TCT CTT GAA GGA GGA GCT CAA TGT GGCGGG-3′ (SEQ ID NO: 14), were inserted into a pSUPER vector(OligoEngine). The resulting plasmid was transfected into 3T3-L1 orDU145 cells with Fugene 6 (Roche). To establish stably transfectedclones, neomycin-derivative G418 (Gibco) was used at a concentration of500 μg/mL, and stable transformants were established. The expressionlevels of the SREBP-1 were evaluated by western blots. For adipogenesisexperiments, 3T3-L1 cells were seeded onto a 96-well plate in a DMEMwith 10% fetal bovine serum and incubated for another two days tocomplete confluence. On day 0, the medium was switched to the inductionmedium: DMEM containing 10% fetal bovine serum, 5 μg/mL of insulin, 0.5mM 1-methyl-3-isobutylxanthin (MIX), and 1 μM dexamethazone (DEX). Onday 2, the induction medium was removed and switched to a DMEM mediumcontaining 10% fetal bovine serum and 5 μg/mL of insulin. On day 10,adipose oil droplets were stained with Oil-Red O. For cell growthexperiments, DU145 cells were seeded onto 96-well plates at density of2,000 cells/well in an MEM without serum or with 1 μg/mL of IGF1, 2%fat-free fetal bovine serum, or 2% fetal bovine serum. The cell growthwas estimated by WST-1 assays after 3 days. The experiments wereperformed in triplicate.

Animal Studies with Fatostatin A

Male mice (129Sv background) were housed under controlled conditions(12-hr light/dark cycle; 25° C.) in the Animal Care Center at BaylorCollege of Medicine and had ad libitum access to standard laboratorychow (Purina Mills) and water. Fatostatin A was administeredintraperitoneally (30 mg/kg; 150 μL) to 5-6 month old male mice (129Svbackground) using two different protocols. First protocol involvesfasting the mice for 48 hrs, followed by refeeding fat free diet foranother 48 hrs. This treatment induces both activities and levels oflipogenic enzymes such as ACC and FAS in addition to SREBPs. Theadministration of fatostatin A or 10% DMSO in PBS to control groups(n=5) started 24 hrs before the fasting and continued daily until theend of the experiment.

In the second protocol, two groups of male mice (n=5) were treated dailyfor two weeks with either 30 mg/kg fatostatin A or 10% DMSO in PBS. Foodintake and body weight were measured daily. At the end of theexperiments, the mice were briefly fasted for 4-5 hrs, and their bloodwas withdrawn for determination of serum constituents. The mice werethen sacrificed, and their livers were quickly removed and ground topowder in liquid nitrogen. The powdered tissues were suspended in 10 mlof PBS containing 0.1 mM PMSF, 5 mM benzamidine, and 5 mg/mL proteaseinhibitor cocktail (Roche), homogenized using Polytron (3×30 Sec, at ahigh speed), and sonicated briefly to degrade DNA. The extracts wereclarified by centrifugation at 16,000×g for 20 min. The samples werethen subjected to western blot analysis using commercially availableantibodies against FAS and SREBP-1. FAS and ACC activities weredetermined as described earlier (Mao et al., 2006).

Example 7 Fatostatin A Prevents Fatty Liver, Reduces Hyperglycemia andInduces Weight Loss in Ob/Ob Mice

Despite the efforts to unravel the networks that regulate food intakeand energy balance, it is not fully understood how obesity causes thesedisease. The effect of fatostatin A on male ob mice was investigated,specifically its effect to prevent weight increase by reducing whiteadipose size, diabetic conditions and fatty liver. As mentioned earlier,fatostatin A is an inhibitor of the master control of transcription byinhibiting the action of SREBP-1. Normal mice treated with fatostatin Alost weight and had lower levels of glucose and cholesterol. FatostatinA reduced the active mature form of SREBP-1 in the liver of treated micecompared to controls.

SREBP-1 and -2 play related but distinct roles in biosynthesis of fattyacids and cholesterol. SREBP-1 preferentially activate the genesrequired for fatty acid synthesis, and SREBP-2 favors cholesterogenesis.Since fatostatin A blocks the activation of SREBP-1 and perhaps SREBP-2,in specific embodiments of the invention administration of fatostatin Ainto obese ob/ob mice transiently modulates the biosynthesis of bothfatty acids and cholesterol and reveals interesting phenotypes in theobese mice.

The Effect of Fatostatin A on Body Weight and Food Intake

The study employed 4-5 week old male ob/ob mice of average weight ofabout 23 g/mouse. Fatostatin A (30 mg/kg/day) was deliveredintraperitoneally daily, and body weight and food intake were measured.As shown in FIGS. 8A-8B, the increase in body weight of the treated micewas significantly lower than the controls. At the end of first week oftreatment ob control mice injected with DMSO gained on average of 4.82g/mouse (from 23.58±0.62 to 28.40±1.45), whereas the fatostatin treatedgroup gained about 3.37 g/mouse (23.08±1.53 to 26.45±1.2 g/mouse),(p=0.03). After 28 days of treatment the fatostatin A treated groupweighed about 12% less than the controls weeks (32.1±1.4 compared to36.2±2.2 g/mouse for the fatostatin) (P=0.02). The accumulative foodintake was similar in both groups FIG. 8C). On average, in the treatedgroup, food intake was not significantly different from the controlsbeing 5.4±1.5 compared to 5.9±1.4 g/mouse·day respectively.

Effect of Fatostatin A on Glucose and Lipids Profile in the Blood

One of the most distinct phenotypes in ob/ob mice is hyperglycemia as aresult of insulin resistance conditions. To determine the effect of thefatostatin A on blood glucose and lipids, the serum levels of glucose,triglycerides, and cholesterol were analyzed in ob/ob mice fed withstandard diet.

As shown in FIGS. 9A-9H, the glucose levels after over night fasting, inserum from treated animals were about 70% lower than the controls;153.2±30.5 and 429.4±87 mg/dl respectively (P=0.003). The glucose levelsin the serum of treated animals became comparable to that of wt micewith functional ob gene, whereas the control mice that were given DMSOwere hyperglycemic as expected. Interestingly, ketone bodies (R-hydroxybutyrate) increased about seven fold in the treated animals compared tothe controls; 3.62±1.41 and 0.5±0.37 mg/dl respectively (P=0.004). Thehigh levels of ketone bodies in fatostatin A animals shows a significantincrease in fatty acid oxidation in livers in which the main product isketone bodies that is secreted in the blood. Also, blood constituentsthat increased in the treated mice were none esterified free fatty acids(NEFA) measured in the serum, which was about 70% higher than that ofthe controls; 1.93±0.26 and 0.7±0.2 mEq/l (P=0.028). This increase inNEFA levels may be due to increased lipolysis from adipose tissue due toan increase in demand for fatty acid oxidation. FFA is known to beassociated with insulin resistance in animals and human. However,despite the elevated levels of FFA in serum of fatostatin A treatedob/ob mice, the glucose level was significantly lower than the controlsindicating an improvement in insulin sensitivity, possibly due toimproved insulin signaling. In addition, it was recently shown that as aconsequence of increased fatty acid oxidation in mouse tissues (liver,adipose and muscle) of mutant acetyl-CoA carboxylase mouse (Acc2^(−/−)mutant mouse), it resulted in higher ketone bodies in the blood andincreased NEFA as a result of increased lipolysis in adipocytes. Thelevel of triglycerides (TG) in the serum increased about 30% in treatedmice compared to controls, 115±11, and 79±12 respectively (P=0.006),indicating that fatostatin A increases secretion and mobilization of TGfrom the liver. The serum level of total cholesterol showed a lowertrend in fatostatin treated animals being 183±16 compared to 219±18mg/dl (P=0.06). However there was a significant decrease of about 35% inLDL (31±3 compared to 48±8; P=0.02) and a lesser decrease in HDL ofabout 22% (144±11 and 183±12; P=0.02). Since there was more decrease inLDL level than that of HDL in serum of fatostatin A treated mice, thisshows a desirable outcome for the treatment with fatostatin A. The levelof VLDL, which transports triglycerides, phospholipids, and cholesteroland is calculated based on TG levels, increased about 50% (23.1±2.3compared to 15.8±2.4 mg/dl).

Fatostatin A Reduces the Size of Epididymal Fat and Ameliorates FattyLiver

Because of uncontrolled food intake, ob/ob mice become morbidly obeseand accumulate excessive levels of fat in fat tissues and in differentorgans, such as liver-causing non-alcoholic fatty liver conditions andinsulin resistance (Hookman and Barkin, 2003). At about 8-9 weeks of agecontrol untreated mice showed enlarged liver size and accumulated fat,as evident from pale color, compared to those treated with fatostatin A(FIG. 10A). The average weight of livers of fatostatin A treated micewas about 32% less than that of the controls (1.59±0.2 compared to2.34±0.15; P=0.06) (FIG. 10D). Liver sections of control mice stainedwith oil red for lipid droplets, contained abundant lipid droplets whilethose of the fatostatin A treated mice were devoid of lipid droplets,which are mainly triglycerides (FIG. 10B). It has been shown thattransgenic mice over expressing SREBP 1 developed fatty liver. However,in ob/ob mice lacking SREBP-1 (lep^(ob/ob)×Srebp 1^(−/−)), fatty liverconditions were significantly improved, suggesting that SREBP 1 is amajor player for the development of fatty liver in ob/ob mice.

At the end of four weeks of fatostatin A treatment the treated miceweighed less than the controls. By examining the epididymal fat pads,which is the major white fat tissue, it was found that the fatostatin Atreated mice has significantly smaller fat pads (FIG. 10C). The averageweight of the fat pads was about 20% less than the controls (2.7±0.1compared to 3.6±0.2; P=0.02) (FIG. 10D). The smaller fat pads may be dueto decrease in storage of lipids and/or decrease in lipogenesis andenhanced fatty acid oxidation in the adipose. Previous studies withAcc2^(−/−) mutant mice showed that the absence of ACC2 also resulted inless fat in livers, smaller epididymal fat pads and enhanced oxidationof fatty acids in different tissues including liver. It was indicatedtherein that enhanced fatty acid oxidation, due to lack of inhibition byACC2-produced malonyl-CoA, on carnitine palmitoyltransferase, the micebecome highly insulin sensitive and protected against diet-inducingobesity and diabetes. In specific aspects, down regulation of ACCenzymes by fatostatin A increases fatty acid oxidation and inhibitsfatty acid synthesis in different tissues such as liver, adipose andmuscle, for example. The TG and cholesterol levels were determined inlivers of fatostatin A treated ob/ob mice and compared to ob/b controls.As shown in FIG. 11A, the TG levels in livers of treated mice werereduced by about 65% (14.8±3.7 and 38.7±6.0 mg/gram liver respectively;P=0.0004). The cholesterol levels in liver were also reduced byfatostatin A by more than 20% (2.8±0.5 and 3.6±0.1; P=0.03) (FIG. 11B).These results further confirm the Oil Red 0 staining and indicate thatfatty liver in ob/ob mice, which is in part caused by increased hepaticlipogenesis, can be completely prevented by treatment with fatostatin A.The reduction of these lipids in liver of treated ob/ob mice is due tosignificant inhibition of lipogenic enzymes needed to synthesize TG andcholesterol or their precursors, in specific aspects of the invention.In addition, due to increased demand for fatty acid oxidation bydifferent mice tissues, including the liver there is an increase inlipase liver activity and also enhanced mobilization of these lipidsfrom liver to the circulation for utilization by different fatty acidoxidizing tissues such as heart and muscles. In specific aspects, thisis related to the higher level of TG in blood of fatostatin A treatedob/ob mice.

Fatostatin A Downregulates Lipogenic Enzymes in Ob/b Mice Liver

Enzymes in lipogenic pathways are regulated by transcription factors,such as PPAR and SREBPs. The effect of fatostatin A on lipogenic enzymeslevels and activities in treated ob/ob mice was examined. The activityof acetyl-CoA carboxylase (ACC), which carries out the rate-limitingstep in fatty acid synthesis, was determined. ACC catalyzes thecarboxylation of acetyl-CoA to yield malonyl-CoA, the building block forfatty acid synthesis, which is carried out by another multifunctionalenzyme, fatty acid synthase (FAS). In addition to the role ofmalonyl-CoA in fatty acid synthesis, it plays an important role in offatty acid oxidation by inhibiting carnitine palmitoyl transferase 1(CPT 1). The lipogenic enzymes are significantly induced in ob/ob mice,partly explaining the morbidly obese phenotype of these mice. Theactivity of ACC in liver extracts of fatostatin A treated mice decreasedby about 40% (3.44±0.44 compared to 5.55±0.57 n mol/min·mg) (FIG. 12A).Fatty acid synthase activity was also significantly downregulated inliver extracts of fatostatin A treated ob/ob mice. FAS activity wasreduced by more than 70% in the treated mice (8.64±1.91 compare to22.6±1.37 n mol/min·mg) (FIG. 12B). The decrease in both ACC and FASactivities is due to reduction in the expression levels for bothenzymes, as shown by western blot analysis for both enzymes (FIG. 12C).Their product fatty acids, C14:0 and C16:0, are significantly lower(about 50%) in the livers of fatostatin treated ob/ob mice than the inthe livers of untreated control mice (Table 2).

ACC is acutely regulated by phosphorylation/dephosphorylation mechanism,resulting in inhibition and activation of the enzyme, respectively. Asshown in FIG. 12C, the level of phosphor-ACC was higher in the controlgroup, however because the expression level of ACC is also higher and tothe same level, this suggests that fatostatin A, does not alter thespecific phosphorylation level (P-ACC/ACC protein). These resultsindicate that the downregulation of ACC activity is solely due todecreased levels of the enzyme and not to a decrease in thephosphorylation status (FIGS. 12C-12D). It was shown previously that inliver there are two ACC isoforms; ACC1 (the dominant isoform in liver)and ACC2 (dominant isoform in muscle) which play distinct roles inregulating lipid synthesis and oxidation, respectively. The reduction inACC and FAS activities indicate that lipogenesis is reduced, whereas fatburning is significantly enhanced in liver of fatostatin A treated mice,which is consistent with the seven fold increase in ketone bodies inblood of ob/ob fatostatin A treated mice as shown in FIGS. 9A-9H. Thelevel of two key enzymes in fatty acid metabolism, which are also undertranscription regulation of SREBP-1, ATP citrate lyase (ACL) andSteroyl-CoA desaturase 1 (SCD1), was determined. The protein level ofACL that converts cytosolic citrate into acetyl-CoA, which is thesubstrate for ACC to yield malonyl-CoA for fatty acid synthesis, wasreduced about 70% in liver extracts of fatostatin A mice. Thisdownregulation in ACL level further amplify the effect of fatostatin Aon the reduction in the lipogenic process in lipogenic tissues such aliver. SCD1 catalyzes the rate-limiting step in the biosynthesis ofmonounsaturated fatty acids, by introducing a Cis double bond in Δ9position of fatty acyl-CoA, such as palmitoyl-CoA and stearoyl-CoA. Theproducts, palmitoleoyl-CoA (16:1) and oleoyl-CoA (18:1) are importantcomponents of triglycerides and cholesterol esters and deletion of SCD1in mice including ob/ob mice resulted in increased metabolic rate,reduced adiposity, preventing fatty liver and protecting againstdiabetes induced by diet. As shown in FIG. 12D, the protein level ofSCD1 was reduced about 50% in liver extracts of fatostatin A treatedmice compared to the controls. This was confirmed by the reduction byabout 70% of the monounsaturated fatty acids C16:1, C18:1 and C20:1 aswell as the reduction by about 50% of the desaturation of theirelongated products (C18:2)N-6, (C18:3)N-6, (C20:2)N-6, and (C20-3)N-6(Table 2). This effect on the reduction in SCD1 is an important factorin weight reduction and decreases TG level in liver and protectionagainst fatty liver, in specific embodiments of the invention.Interestingly, the protein level of FADS 1 or Δ5 Desaturase did notchange as a result of fatostatin A treatment.

TABLE 2 Gas Chromatography-Mass Spectrometry (GC-MS) Analyses Of FattyAcids in the Livers of OB/OB Treated Mice and Their Untreated ControlFatostatin A Control Ratio Fatty Acid μmole/gram liver μmole/gram livertreated:control P value (14:0) 1224.567 ± 508.88  2378.586 ± 329.454 0.51483 0.047781 (16:0) 37143.23 ± 3656.16  85044.75 ± 5716.175 0.4367490.001619 (18:0) 15067.14 ± 916.48   14129.2 ± 272.7029 1.066383 0.260833(20:0) 164.3537 ± 22.89   192.9773 ± 16.46977 0.851674 0.059842 (22:0)116.1305 ± 25.97   91.74762 ± 12.60782 1.26576 0.2499 (24:0) 157.8261 ±7.02   120.6449 ± 19.37201 1.308188 0.052305 (16:1) 11429.29 ± 227.5  34787.26 ± 4482.311 0.328548 0.005542 (18:1) 44471.72 ± 8840.37 152218.4 ± 12872.57 0.292157 0.001621 (20:1) 593.7212 ± 119.68  1829.537± 230.8927 0.32452 0.00368 (22:1) 94.94588 ± 14.91   109.0117 ± 11.474130.870969 0.379216 (24:1) 397.92 ± 216.3  234.2073 ± 26.9417  1.6990080.214302 (18:2)N-6 29673.93 ± 2456.22   46513.4 ± 2825.032 0.6379650.00455 (18:3)N-6 237.1671 ± 40.5   560.4358 ± 61.89641 0.423183 0.00546(20:2)N-6 426.6694 ± 63.52   914.0388 ± 90.59441 0.466796 0.001424(20:3)N-6 1630.394 ± 193.18  2589.236 ± 154.791  0.629682 0.008568(20:4)N-6 8747.289 ± 781.41  7772.095 ± 110.7878 1.125474 0.171108(22:4)N-6 280.2708 ± 46.1   292.2569 ± 11.81504 0.958988 0.810355(22:5)N-6 172.9715 ± 64.89   146.5646 ± 14.54166 1.180173 0.879433(18:3)N-3 4416.975 ± 643.65  83625.879 ± 220.5747  1.21818 0.703923(20:5)N-3 2145.643 ± 265.17  3067.856 ± 299.7701 0.699395 0.04467(22:5)N-3 1720.424 ± 221.16  2338.451 ± 234.2185 0.735711 0.092988(22:6)N-3 9223.38 ± 700.31  8718.014 ± 532.5817 1.057968 0.448218

Liver samples (100 mg) obtained from fatostatin A-treated ob/ob mice andnon-treated control mice and stored at −80° C. until analyzed for fattyacid contents. The fatty acids were extracted according to Folch'sprotocol and quantitatively analyzed using gas chromatography-massspectrometry (GC-SM). As shown in the above table, there is about 50%reduction of C14:0 and C16:0, the products of the de novo fatty acidsynthesis, and about 70% reduction of monounsaturated fatty acids C16:1,C18:1 and C20:1 and their elongation-desaturation products (C18:2)N-6,(C18:3)N-6, (C20:2)N-6, (C20-3)N-6 and (C20:5)N-3. Myristate (C14:0) andpalmitate (C16:0), the products of FAS, were reduced by about 50%(P<0.05). There was no change in C18 levels, which is derived not onlyfrom de novo fatty acid synthesis by FAS, but also from food and chainelongation system. Interestingly there was a strong trend in loweringthe C20:0 by about 15% (P+0.059) and an increase of 30% in C24:0(P=0.05). In parallel to the significant decreases in long-chainsaturated fatty acids, results show that there were significantreductions of about 70% in the levels of monounsaturated fatty acidsC16:1, C18:1 and C20:1 (P<0.004). Also, the levels of polyunsaturatedlong-chain fatty acids (18:2)N-6, (18:3)N-6, (20:2)N-6, (20:3)N-6 and(20:5)N-3 were reduced 30-60%. These reductions are the result of downregulation of key enzymes in the lipogenic pathways (FAS, ACC and SCD,ACL) at transcription and translation. These results help explain theeffect of fatostatin A in amelioriating fatty liver conditions byreducing the triglyceride levels made in the liver.

Downregulation of mRNA Levels of Lipogenic Enzymes

The decrease in protein levels can be attributed to a transcriptional ortranslational regulation. Real time PCR was used to determined thelevels of mRNA levels of representatives of lipogenic genes ACC1, FASand SCD1 in addition to the lipogenic transcription factor PPAR γ. Therewas about 80% reduction in the mRNA levels of ACC1, FAS and SCD1 (FIG.13). These results are consistent with lower levels of enzyme proteinsand activities, and strongly indicate that fatostatin A lowerslipogenesis by inhibiting the maturation of SREBP-1. The down regulationof lipogenic enzyme involves one of its main transcription factors, PPARγ, in specific embodiments. The mRNA level of this transcription factorwas reduced by about 40% in extracts of fatostatin A treated mice (FIG.13). Since, ob/ob mice treated with fatostatin A reduced hyperglycemiaand prevented fatty liver, this indicates that PPAR γ in liver is one ofseveral factors that may affect these pathological conditions, inspecific aspects. In summary, Fatostatin A through its action on SREBP-1ameliorated fatty liver by reducing hepatic TG storage, reducedadiposity and lowered hyperglycemia in treated ob/ob mice. These studiesindicate that fatostatin A and its analogs are useful agents againstobesity, fatty liver and diabetes, for example.

Four-to 5-week old homozygous male obese (ob/ob) mice (C57BL/6J, TheJackson Laboratory, Bar Harbor, Me.) were housed under controlledconditions (12-hr light/dark cycle; 25° C.). The animals were housed 5per cage and had ad libitum access to standard laboratory chow (PurinaMills, Richmond Ind.) and water for one week after their arrival. Onfirst day of the experiment and every day thereafter the weight of themice and the amount of the food consumed were measured. The weight ofmice and food remaining were measured daily between 3-5 p.m. before theip injection of fatostatin A (30 mg/kg; 150 μL). The administration ofFatostatin A or 10% DMSO in PBS to control groups (n=5) continued dailyfor four weeks till the end of the study.

After 28 days of daily injection of fatostatin A mice were fastedovernight and blood was withdrawn and Whole blood glucose and3-hydroxybutyrate were measured with a Glucometer Precision Xtra(Abbott). For determination of serum constituents. Glucose, triglycerideand cholesterol measurements were done by the Comparative PathologyLaboratory (Baylor College of Medicine). Serum non-esterified fattyacids (NEFA) were measured by using NEFA C kit (Wako Chemicals,Richmond, Va.).

Mice were sacrificed and weights of livers, and Epididymal fat pads weredetermined. Frozen sections of Liver slices from individual animals werestained with Oil Red 0 to visualize the fat droplets (TG) in liverslices as described earlier (Abu-Elheiga et al., 2001). The remainingliver tissues were frozen in liquid nitrogen and kept at −80° C. forfurther analysis.

Liver triglyceride and cholesterol contents were carried out asdescribed in the reference (Chandler, et al., 2003) using Cholesterol EKit (Wako) and Infinity Triglyceride Kit (Thermo Electron, Melbourne,Australia), adapted for colorimetric analysis in 96-well plate format.

Enzymatic Activities and Western Blot Analyses

A portion of the frozen liver was ground to powder in liquid nitrogen.The powdered tissues were suspended in 10 ml of PBS containing 0.1 mMPMSF, 5 mM benzamidine, and 5 mg/ml protease inhibitor cocktail (Roche),and homogenized using Polytron (3×30 Sec, at high speed) and sonicatedbriefly to degrade DNA. The extracts were clarified by centrifugation at16,000×g for 20 min. Protein concentrations in the supernatant weredetermined, and subjected to western blot analysis using commerciallyavailable antibodies against the following enzymes: FAS (BDBiosciences), citrate lyase SCD1, FADS1, ACC and phospho-ACC antibodies.The proteins were visualized using Amersham ECL Plus™ Western BlottingDetection Reagents. The intensity of the specific bands of proteins ofinterest were scanned and normalized against beta-actin forquantifications. FAS and ACC activities from the liver extracts weredetermined as described earlier (Mao et al., 2006).

Quantitative Real Time PCR

Total RNA was prepared from mouse tissues using TRIzol reagent(Invitrogen). Equal amounts of RNA from 5 mice were pooled and treatedwith DNase I (Turbo DNA-free, Ambion, Inc.). First stranded cDNA wassynthesized from 2 μg of DNase I-treated total RNA with random hexamerprimer using Superscript II RNase H-reverse transcriptase (Invitrogen).The real time PCR contained, in a final volume of 20 μl, 10 ng ofreverse transcribed total RNA, 0.5 μM forward and reverse primers, and10 μl of 2× master mix from DyNAmo HS SYBR Green qPCR kit (Finnzymes).PCR was carried out in 96-well plate using DNA Engine Opticon System (MJResearch, Inc). All reactions were done in triplicate and the relativeamounts of mRNAs were calculated using the comparative C(t) method. Thecycle threshold C(t) was calculated using the Opticon Monitor software2.02 (MJ Research). Mouse β-actin mRNA was used as the internal control.Data were expressed as the mean±SD. Difference between two groups wasassessed using the unpaired two-tailed Student t-test.

Example 8 Identification of Target Molecules of Fatostatin A and Analogsor Derivatives Thereof

In certain aspects of the invention, one or more targets of fatostatin Aor its analog or derivative is identified. Although any suitable methodmay be employed for such identification, in specific embodiments thefatostatin A or analog or derivative thereof, is labeled. Exemplarylabels include biotin, for example.

Example 9 Exemplary Compounds and Modifications Thereof

FIGS. 14A-14F illustrate exemplary compounds of the invention, and theirgiven names are provided in Table 3 and Table 4. FIGS. 15-17 demonstrateexemplary luciferase reporter gene assays for these exemplary compoundsat 20 mM by the same method shown in FIG. 2A. The adipogenesis assay wasperformed as described (Choi et al., 2003). The analogues thatcompletely inhibited the formation of oil droplets in cells were scoredto be adipogenesis-inhibiting analogues.

TABLE 3 Exemplary Compounds of the Invention Inhibition of Name entryLuc/Gal STDEV adipogenesis none 9.4426 1.05772-propyl-4-(4-p-tolylthiazol-2-yl)pyridine  1 6.2297 1.1014 +4-(4-(4-bromophenyl)thiazol-2-yl)-2-  2 4.5130 0.6176 − propylpyridine4-(4-phenylthiazol-2-yl)-2-propylpyridine  3 7.4643 2.2215 −4-(4-(4-chlorophenyl)thiazol-2-yl)-2-  4 6.3808 2.0425 − propylpyridine4-(4-(4-ethylphenyl)thiazol-2-yl)pyridine  5 7.6190 1.6221 −4-(4-p-tolylthiazol-2-yl)pyridine  6 13.4689 1.6735 −4-(4-(4-methoxyphenyl)thiazol-2-yl)pyridine  7 18.3174 2.9172 −4-(2-(2-propylpyridin-4-yl)thiazol-4-  8 7.7585 1.6193 +yl)phenylbenzoate 4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenol  914.5234 2.7276 + methyl 2-(4-(2-(2-propylpyridin-4-yl)thiazol-4- 1010.7717 0.8662 − yl)phenoxy)acetate 4-(4-chlorophenyl)-2-(3,4- 12 5.92141.2693 − dimethoxyphenyl)thiazole4-(4-(3,4-dichlorophenyl)thiazol-2-yl)-2- 13 5.2391 0.4021 −propylpyridine 4-(4-(4-fluorophenyl)thiazol-2-yl)-2- 14 9.6605 0.9824 −propylpyridine 4-(4-(2,4-difluorophenyl)thiazol-2-yl)-2- 15 10.83831.7661 − propylpyridine 4-(2-(2-propylpyridin-4-yl)thiazol-4- 16 10.33382.0763 + yl)benzenamine N-isopropyl-4-(2-(2-propylpyridin-4-yl)thiazol-17 4.7079 1.2781 + 4-yl)benzenamineN-(4-(2-(2-propylpyridin-4-yl)thiazol-4- 18 11.7685 6.8358 +yl)phenyl)acetamide None 15.0759 0.83052-propyl-4-(4-p-tolylthiazol-2-yl)pyridine  1 7.5537 0.9784 +N-(4-(2-(2-propylpyridin-4-yl)thiazol-4- 19 4.1981 0.4653 +yl)phenyl)methanesulfonamideN-benzyl-4-(2-(2-propylpyridin-4-yl)thiazol-4- 20 5.6748 0.0613 +yl)benzenamine N-(cyclopropylmethyl)-4-(2-(2-propylpyridin-4- 21 6.43781.2736 + yl)thiazol-4-yl)benzenamine4-(4-bromophenyl)-2-(2-propylpyridin-4- 22 21.5911 0.8383 −yl)thiazole-5-carboxylic acid methyl4-(4-bromophenyl)-2-(2-propylpyridin- 23 8.1137 2.5369 +4-yl)thiazole-5-carboxylate 4-(4-(4-methoxyphenyl)thiazol-2-yl)-2- 2411.0367 2.1112 − propylpyridine 4-(4-(3-methoxyphenyl)thiazol-2-yl)-2-25 7.8536 1.2799 propylpyridine 4-(4-(2-methoxyphenyl)thiazol-2-yl)-2-26 8.3046 2.6780 + propylpyridine 2-phenyl-4-p-tolylthiazole 27 13.82221.3938 − 3-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenol 28 12.77911.1429 − 2-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenol 29 8.2379 1.9501− 4-(4-bromophenyl)-N-isopropyl-2-(2- 30 16.0226 2.1917 −propylpyridin-4-yl)thiazole-5-carboxamide4-(4-(4-chlorophenyl)thiazol-2-yl)pyridine 31 16.9971 2.6512 −4-(4-(4-chlorophenyl )thiazol-2-y l)-2- 32 9.5798 0.8524 ethylpyridine4-(4-chlorophenyl)-2-phenylthiazole 33 17.8175 3.7158 −2-propyl-4-(4-(thiophen-2-yl)thiazol-2- 34 12.1593 1.5587 + yl)pyridine2-phenyl-4-p-tolylthiazole 27 13.8222 1.3938 − None 18 22-propyl-4-(4-p-tolylthiazol-2-yl)pyridine  1 9.413845 0.840651 +4-(4′-methyl[1,1′-biphenyl]-4-yl)-2- 35 11.35866 0.881475 + propyl)pyridine 2-(2-propylpyridin-4-yl)-4-p-tolylthiazole-5- 36 18.988892.082093 carboxylic acid 2-ethyl-4-(4-p-tolylthiazol-2-yl)pyridine 379.869906 0.71108 4-phenyl-2-(2-propylpyridin-4-yl)thiazole-5- 3822.65811 3.898667 carboxylic acid methyl 2-(2-propylpyridin-4-yl)-4-p-39 14.92978 2.600443 tolylthiazole-5-carboxylate None 8.2181 0.50972-propyl-4-(4-p-tolylthiazol-2-yl)pyridine  1 3.4437 0.2720 tert-butyl4-(2-(2-propylpyridin-4-yl)thiazol-4- 40 2.4390 0.4730yl)phenylcarbamate N-cyclohexyl-4-(2-(2-propylpyridin-4- 41 6.52290.8638 yl)thiazol-4-yl)benzenamine4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)-N- 42 3.3957 0.3619tosylbenzenamine N-(4-(2-(2-propylpyridin-4-yl)thiazol-4- 43 2.85060.6396 yl)phenyl)-8-quinolinesulfonamideN-(4-(2-(2-propylpyridin-4-yl)thiazol-4- 44 1.8538 0.1240yl)phenyl)-2-thiophenesulfonamide

TABLE 4 Name Compound 4-(4-bromophenyl)-2-(pyrrolidin-2-yl)thiazole 454-(4-bromophenyl)-2-(1-propylpyrrolidin-2-yl)thiazole 46 tert-butyl2-(4-(4-bromophenyl)thiazol-2- 47 yl)pyrrolidine-1-carboxylate benzyl2-(4-(4-bromophenyl)thiazol-2- 48 yl)pyrrolidine-1-carboxylate3-(4-(4-bromophenyl)thiazol-2-yl)-1-propylpiperidine 49 tert-butyl3-(4-(4-bromophenyl)thiazol-2- 50 yl)piperidine-1-carboxylate benzyl3-(4-(4-bromophenyl)thiazol-2- 51 yl)piperidine-1-carboxylate benzyl4-(4-(4-bromophenyl)thiazol-2- 52 yl)piperidine-1-carboxylate benzyl(R)-2-(4-(4-(methylsulfonamido)phenyl)thiazol-2- 53yl)pyrrolidine-1-carboxylate benzyl3-(4-(4-(methylsulfonamido)phenyl)thiazol-2- 54yl)piperidine-1-carboxylate benzyl4-(4-(4-(methylsulfonamido)phenyl)thiazol-2- 55yl)piperidine-1-carboxylate4-(3-(pyridin-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)- 56N-tosylbenzenamine 3-chloro-4-methyl-N-(6-(4-(3-(trifluoromethyl)ben- 57zyl)piperazin-1-yl)pyridin-3-yl)benzenesulfonamide(4-(5-chloro-2-methylphenyl)piperazin-1-yl)(4- 58(tosylamino)phenyl)methanone 4-(4-((1-methyl-1H-benzo[d]imidazole-2- 59yl)methyl)piperazin-1-yl)-N-tosylbenzenamine4-chloro-N-(4-(4-((1-methyl-1H-benzo[d]imidazol-2- 60yl)methyl)piperazin-1-yl)phenyl)benzenesulfonamide(Z)-4-(3-cyano-3-(4-(2,4-dimethylphenyl)thiazol-2- 61yl)allyl)-N-(thiazol-2-yl)benzenesulfonamideN-(3-(H-imidazo[1,2-a]pyridine-2-yl)phenyl)-4-methyl- 622-phenylthiazole-5-carboxamideN-(3-(benzo[d]thiazol-2-yl)phenyl)isonicotinamide 633-(4-chlorophenyl)-4,5-dihydro-1-phenyl-5-(2- 64phenylthiazol-4-yl)-1H-pyrazoleN-(4-(6-methylbenzo[d]thiazol-2-yl)phenyl)-2-(N- 65m-tolylmethylsulfonamido) acetamideN-(4-(6-methylbenzo[d]thiazol-2-yl)phenyl)-2-(N- 66p-tolylmethylsulfonamido)acetamide

Furthermore, a skilled artisan recognizes that it may be suitable tomodify one or more aspects of an exemplary compound to assist inidentifying other suitable compounds. For example, upon determination ofsuitability for a particular compound for treatment and/or prevention ofone or more metabolic disorders, the compound may be modified toidentify other related compounds for use for the same or a differentmetabolic disorder. Such alterations may occur in accordance withexemplary chemical groups as described herein, in specific embodiments.

Example 10 Blockage of Fat Synthesis by Inhibiting the Activation ofSREBP

Upon fat depletion in a cell, sterol regulatory element binding proteins(SREBPs) are released proteolytically from the membrane and translocatedinto the nucleus, where they activate transcription of the genesinvolved in cholesterol and fatty acid biosynthesis. In the presentinvention, it is shown that a small synthetic molecule that blocksadipogenesis is a selective inhibitor of the SREBP activation. Thediarylthiazole derivative, called fatostatin, impairs the proteolyticactivation of SREBPs, thereby decreasing the transcription of lipogenicgenes in cells. The molecular target of fatostatin appears to be SREBPcleavage-activating protein (SCAP). Fatostatin blocked increases in bodyweight, blood glucose, and hepatic fat accumulation in obese ob/ob mice,even under uncontrolled food intake.

As described herein, fatostatin inhibits the insulin-inducedadipogenesis of 3T3-L1 cells and the serum-independent growth of DU145cells (Choi et al., 2003). Gene expression profiles of drug-treated anduntreated cells were compared to gain information about specificmolecular pathways affected by fatostatin. DU145 cells were treated withfatostatin or DMSO alone, and the extracted mRNA samples were analyzedby Affymetrix DNA microarrays mapping 33,000 genes. Of those genes (allof which are available at the National Center for BiotechnologyInformation's GenBank database on the world wide web), transcriptionlevels of 63 genes decreased at least 35% in response to fatostatintreatment (Table 5). Thirty-four of the affected genes were directlyassociated with fat or sterol synthesis, such as genes encodingbiosynthetic enzymes, and 18 of the affected genes have been reported tobe controlled by SREBPs (Horton et al., 2003). Downregulation of theaffected SREBP-responsive genes was confirmed by RT-PCR experiments. Thehigh occurrence of the SREBP-responsive genes and fat/cholesterolbiosynthesis genes in the list of the downregulated genes implies thatfatostatin acts on the SREBP pathway.

Table 5 shows the results of the microarray analysis. The 18 underlinedgenes have been reported to be controlled by SREBPs, the genes in boldare associated with fat or sterol synthesis.

TABLE 5 P D 0.707107 Hs.17414 NM_00109ATP citrate lyase/FL = gb:NM_001096.1 P D 0.707107 Hs.11223 NM_00589Isocitrate dehydrogenase 1 (NADP+), soluble/FL = gb:AF020038.1gb:AF113917 P D 0.707107 Hs.268012 D89053.1 Fatty-acid-Coenzyme Aligase, long-chain 3/FL = gb:NM_004457.2 gbz:D89053.1 gb:AF116690.1 P D0.707107 Hs.155976 AI670819 Cullin 4B/FL = gb:NM_003588.1 gb:AB014595.1P D 0.707107 Hs.41693 BG252490 DNAJ (Hsp40) homolog, subfamily B, member4/FL = gb:U40992.2 gb:NM_007034.2 P D 0.707107 Hs.14732 AL049699malic enzyme 1, NADP(+)-dependent, cytosolic/FL = gb:NM_002395.2 P D0.707107 Hs.213289 S70123.1 low density lipoprotein receptor (familialhypercholesterolemia) P D 0.707107 Hs.6986 AL565516 human glucosetransporter pseudogene P D 0.659754 Hs.75616 NM_014676 seladin-1/FL =gb:AF261758.1 gb:BC004375.1 gb:NM_014762.1 P D 0.659754 Hs.77393NM_00200 farnesyl diphosphate synthase (farnesylpyrophosphate synthetase, dimethylally-transtransferase, geranyltranstransferase P D 0.659754 Hs.132898NM_00593 fatty acid desaturase 1 P D 0.659754 Hs.274398 BC002654 Homosapiens, similar to tubulin, beta4, clone MGC:4083, mRNA, complete cds PD 0.659754 BC005838 Homo sapiens, tubulin, beta5 P D 0.659754 Hs.115285BF978872 dihydrolipoamide S-acetyltransferase (E2 component of pyruvatedehydrogenase complex) P D 0.659754 Hs.159154 AL565749 Tubulin, beta 4 PD 0.659754 Hs.179817 AF167438 Homo sapiens androgen-regulated short-chain dehydrogenasereductase 1 (ARSDR1) P D 0.615572 Hs.213289 NM_00052low density lipoprotein receptor (familialhypercholesterolemia)/FL = gb:NM_000527.2 P D 0.615572 Hs.75105 NM_00657emopamil-binding protein (sterol isomerase)/ FL = gb:NM_006579.1 P D0.615572 Hs.74304 NM_00270 Homo sapiens periplakin (PPL) A D 0.615572Hs.268515 NM_00243 meningioma (disrupted in balanced translocation) 1/FL= gb:D63807.1 P D 0.615572 UG = Hs.31 AF096304 Homo sapiens putativesterol reductanse SR-1 (TM7SF2) transmembrane 7 super family 2/FL =gb:AF096304.1 P D 0.615572 Hs.93199 D63807.1 lanosterol synthase(2,3-oxidosqualene- lanosterol cyclase)/FL = gb:D63807.1 P D 0.574349Hs.119597 AB032261 stearoyl-CoA desaturase (delta-9-desaturase)/FL = gb:AF097514.1 gb:NM_005063 gbL/ab932261.1 P D 0.574349Hs.174140 AI971281 ATP citrate lyase/FL = gb:NM_001096.1 P D 0.574349Hs.171825 NM_00367 basic heliz-loop-helix domain containing, class B,2/FL = gb:AB004066.1 gb:NM_003670.1 P D 0.574349 Hs.79103 AW235051cytochrome b5 outer mitochondrial membrane precursor/FL = gb:BC004373.1gb:NM_030579.1 P D 0.574349 Hs.11806 NM_00136 dehydrocholesterolreductase/ FL = gb:BC000054.1 gb:AF034544.1 gb:AF067127.1 gb:AF096305gb:NM_001360.1 P D 0.574349 Hs.3838 NM_00662 serum-inducible kinase/FL =gb:AF059617.1 gb:NM_006622.1 gb:AF223574.1 P D 0.574349 Hs.213289AI861942 low density lipoprotein receptor (familialhypercholesterolemia)/FL = gb:NM_000527.2 P D 0.574349 Hs.92199 AW084510lanosterol synthase (2,3-oxidosqualene- lanosterol cyclase)/FL =gb:NM_002340.1 gb:NM_U22526.1 P D 0.574349 Hs.5920 NM_00547UDP-N-acetylglucosamine-2-epimerase-N- acetrylmannosamine kinase P D0.574349 Hs.1524 NM_00381 tumor necrosis factor (ligand) superfamily,member9/FL = gb:NM_003811.1 gb:U03308.1 P D 0.574349 Hs.278544 BC000408acetyl-Coenzyme A acetyltransferase 2 (acetoacetyl Coenzyme Athiolase)/FL = gb: BC000408.1 P D 0.574349 Hs.174140 U18197.1ATP citrate lyase/FL = gb:U18197.1 P D 0.574349 Hs.7232 B3855983acetyl-Coenzyme A carboxylase alpha/ FL = gb:NM_000664.1 gb:U19822.1 P D0.535887 Hs.226213 NM_00078 cytochrome P450, 51 (lanosterol 14-alpha-demthylase)/FL= gb:U23042.1 gb:NM_000786 gb:D55653.1 A D 0.535887Hs.268490 NM_00047 nuclear receptor subfamily O, group B, member 1/FL =gb:NM_000475.2 P D 0.535887 Hs.48876/ :AA872727 famesyl-diphosphatefarnesyltransferase/ FL = gb:L06070.1 gb:L06105.1 gb:NM_004462.1 P D0.535887 Hs.65270 NM_00603 lipase, endothelial/FL = gb:AF118767.1gb:NM_006033.1 0.535887 Hs.14779 AK000162 acetyl-CoA synthetase 0.535887Hs.44499 U59479.1 pinin, desmosome associated protein/DEF = Humanneutrophil protein mRNA, partial cds P D 0.5 Hs.154654 AU144855cytochrome P450, subfamily I (dioxin- inducible), polypeptide 1(glaucoma 3, primary infantile) P D 0.5 Hs.11899 AL5186273-hydroxy-3-methylglutaryl-Coenzyme A reductase/FL = gb:M11058.1gb:NM_000859.1 0.5 Hs.2178 NM_00352 H2B histone family, member Q/FL =gb: NM_003528.1 P D 0.5 Hs.130607 NM_00043mevalonate kinase (mevalonic aciduria)/ FL = gb:M88468.1 gbz:NM_000431.1P D 0.5 Hs.284244 NM_00200 fibroblast growth factor 2 (basic)/FL = gb:M27968.1gb:NM_002006.1 P D 0.5 Hs.71465 AF098865squalene epoxidase/FL = gb:D78130.1 gb:AF098865.1 gb:NM_003129.2 P D 0.5Hs.288031 D85181.1 sterol-C5-desaturase (fungal ERG3, delta-5-desaturase)-like/FL = gb:D85181.1 A D 0.5 L32662.1 prostaglandin E2receptor/DB_XREF = gi: 484163/FL = gb:L32662.1 P D 0.5 Hs.81412 D80010.1lipin 1 P D 0.466517 Hs.11899 NM_000853-hydroxy-3-methylglutaryl-Coenzyme Areductase/FL = gb:M11058.1 gb:NM_000859.1 P D 0.466517 Hs.79440 NM_00654IGF-II mRNA-binding protein 3/FL = gb: U07188.1 gb:U76705.1gb:AF117108.1 gb:NM_006547.1 P D 0.466517 Hs.75318 AL575074 tubulin,alpha 1 (testis specific) P D 0.466517 Hs.226213 U40053cytochrome P450, 51 (lanosterol 14-alpha- demethylase) P D 0.466517Hs.187579 NM_01637 hydroxysteroid (17-beta) dehydrogenase 7/ FL =gb:AF098786.2 gb:NM_016371.1 P D 0.466517 Hs.76038 BC005247isopentenyl-diphosphate delta isomerase/ FL = gb:BC005247.1 P D 0.466517Hs.57698 BC000245 NAD(P) dependent steroid dehydrogenase- like;H105e3/FL = gb:BC000245.1 gb:U4710 gb:NM_015922.1 P D 0.353553 Hs.77910NM_00213 3-hydroxy-3-methylglutaryl-Coenzyme asynthase 1 (soluble)/FL = gb:BC000297.1 gb:L25798.1 gb:NM_015922.1 P D0.329877 Hs.56205 BE300521 insulin induced gene 1/FL = gb:NM_005542.1 PD 0.329877 Hs.154654 NM_00010 cytochrome P450, subfamily I (dioxin-inducible), polypeptide 1 (glaucoma3, primary infantile)/FL =gb:NM_000104.2 gb:U0 P D 0.329877 Hs.3828 A1189359mevalonate (diphospho) decarboxylase/ FL = gb:U49260.1 gb:CB000011.1gb:NM_002461.1

To confirm that fatostatin impairs the function of SREBPs, the abilityof endogenous SREBPs to activate transcription of an SREBP-responsivereporter gene was measured in CHO-K1 cells in the presence or absence offatostatin (FIG. 18A). Fatostatin decreased activation of the reportergene, in which the expression of luciferase is controlled by sterolregulatory elements. Fatostatin had limited effect on the ability of anexogenously expressed, mature form of SREBP-1 (amino acids 1-436) toactivate the reporter gene (FIG. 18B), indicating that fatostatinimpairs the activation process of SREBPs.

To determine if fatostatin affects the ER-Golgi translocation andproteolytic processing of SREBPs, a reporter assay developed by Sakai etal. (1998) was used. PLAP-BP2 in transfected CHO-K1 cells remainsmembrane-bound unless it is cleaved by S1P in the Golgi and secretedinto the culture medium. In the assay, a secreted alkaline phosphatase,fused with an SREBP-2 fragment lacking the NH2-terminal DNA-bindingdomain (PLAP-BP2513-1141), permits monitoring of translocation andprocessing through changes in the fluorescence of a fluorogenicphosphatase substrate (FIG. 18C). When cells were co-transfected withplasmids encoding PLAP-BP2513-1141 and SCAP, PLAP phosphatase wassecreted, generating fluorescence signals. Secretion was similarlydecreased by the addition of fatostatin or sterols (FIG. 18C). Thefatostatin-mediated inhibition of SREBP activation was confirmed bywestern blot analysis of SREBPs. Treatment of the CHO-K1 cells withfatostatin decreased the amount of the 68 KDa mature form of SREBP-2,and increased the amount of the 125 KDa precursor form (FIG. 18D).Similar results were obtained for SREBP-1 (FIG. 22). These resultscollectively indicate that fatostatin blocks the activation process ofboth isoforms of SREBP.

The inventors considered that fatostatin impairs either the proteolyticcleavage of SREBPs in the Golgi apparatus or the ER-to-Golgitranslocation of the SCAP/SREBP complex. Brefeldin A, a natural productthat blocks anterograde movement of proteins from the ER to the Golgi,is known to render SREBPs unresponsive to sterols, and causes SREBPs tobe constitutively processed in the ER by relocating S1P from the Golgito the ER (DeBose-Boyd et al., 1999). In the presence of brefeldin A,fatostatin had no impact on the SREBP processing (FIG. 19A), suggestingthat fatostatin does not block the proteolysis itself.

To determine whether fatostatin blocks the ER-to-Golgi translocation ofthe SCAP/SREBP complex, the inventors analyzed the extent of N-linkedglycosylation of SCAP in the Golgi apparatus. SCAP contains aglycosylated luminal loop that is protected from proteolysis by trypsinand recognizes anti-SCAP IgG-9D5. The two oligosaccharides in the loopare sensitive to endoglycosidase H when SCAP resides in the ER. As SCAPis transported to the Golgi, its sugars become resistant to digestion byendoglycosidase H. The translocated SCAP has higher levels ofglycosylation, and is more resistant to endoglycosidase H than ER-boundSCAP. Sterols prevent SCAP from becoming resistant to endoglycosidase Hby inhibiting the ER-Golgi translocation (Nohturfft et al., 1998). Cellswere grown in the absence or presence of fatostatin or sterols, andmembrane fractions were treated successively with trypsin andendoglycosidase H. In cells grown without fatostatin and sterols, atryptic fragment of SCAP was more resistant to endoglycosidase H and hadone or two saccharide chains (FIG. 19B, lane 1). When cells were grownin the presence of fatostatin or sterols, the SCAP fragment was lessresistant to endoglycosidase H, and had either zero or one saccharidechain (FIG. 19B, lanes 2 and 3). Thus fatostatin appears to inhibit thetranslocation of SCAP from the ER to the Golgi.

Studies of the structure-activity relationship of fatostatin indicatedthat the molecule retains or even increases biological activity when itstoluene moiety is modified with a variety of alkyl or aryl sulfonamidegroups. One fluorescent derivative, dansyl fatostatin (FIG. 20A),retained the ability to block SREBP activation (FIG. 24) and served as amicroscopic probe. Confocal microscopic analyses revealed that thelocalization of dansyl fatostatin overlapped with that of ER-trackerred, a specific marker for ER (FIG. 20B). In contrast, the controldansyl molecule, which lacked fatostatin, failed to localize to anyorganelle. The selective ER localization implies that fatostatin bindsto a protein in the ER; the most likely candidate is SCAP, the target ofcholesterol for the control of SREBP (Radhakrishnan et al., 2004). Totest this hypothesis, proteins bound to a fatostatin-polyprolinelinker-biotin conjugate (FIG. 20A) (Sato et al., 2007) were purifiedfrom cell lysates and analyzed by western blots with antibodies againstSCAP, SREBP-1, SREBP-2, and ATF6, an unrelated ER-bound transcriptionfactor (Ye et al., 2000). The results showed that fatostatin was boundto SCAP, but not to the other proteins (FIG. 20C). The binding was lostupon addition of excess fatostatin, but not excess cholesterol (FIG.20D), raising the possibility that fatostatin may interact with SCAP ina site distinct from that of cholesterol.

Having established a key role of SREBPs in lipogenesis, thepharmacological effects of fatostatin on ob/ob mice, a mouse model ofobesity with uncontrolled food intake, was then examined. Fatostatin wasdelivered intraperitoneally on a daily basis, and food intake and bodyweight were monitored. The average daily food intake by the treated micewas not significantly different from that of the controls (5.4+1.5 vs.5.9+1.4 g/mouse/d, respectively, p>0.05), and no obvious toxicity wasobserved during the treatment. After 28 days of treatment withfatostatin, the treated mice weighed about 12% less than the untreatedcontrols (32.1±1.4 and 36.22 g/mouse, respectively, p=0.02). One of themost distinct phenotypes in ob/ob mice is hyperglycemia resulting frominsulin resistance. Examination of blood constituents revealed that theaverage glucose level of the treated mice was ˜70% lower than that ofthe untreated mice (153.2±30.5 vs. 429.4±87 mg/dl, respectively,p=0.003), which is in the range of normal glucose levels. These resultsare consistent with the reported role of SREBP-1c in the pathogenesis ofhepatic insulin resistance Ode et al., 2004).

Another phenotype of ob/ob mice is excessive accumulation of fat inorgans, including non-alcoholic fatty liver. Enlarged and fatty liverswere evident from their pale color in the untreated ob/ob mice, whilelivers of the mice treated with fatostatin appeared normal. Livers ofthe treated mice averaged ˜32% less weight, and fat pads were smallerthan those of the untreated mice. Oil red staining of the liver sectionsshowed that the livers of the untreated ob/ob mice contained abundantlipid droplets, while livers of the treated mice contain lower levels oflipid accumulation (FIG. 21). The triglyceride and cholesterol levels inthe livers of the treated mice were also reduced. The prevention offatty liver in ob/ob mice by fatostatin is in agreement with thereported role of SREBP-1 in developing fatty liver: transgenic miceoverexpressing SREBP-1 developed fatty livers, while ob/ob mice lackingSREBP-1 (lep^(ob/ob)×Srebp 1^(−/−)) had healthy livers (Yahagi et al.,2002).

The reduction of hepatic fat levels in the treated mice was thought tobe due to decreased hepatic expression of SREBP-responsive lipogenicenzymes. Therefore, the effects of fatostatin were examined on thehepatic protein levels and enzymatic activities of representativeSREBP-responsive lipogenic enzymes, including fatty acid synthase (FAS),acetyl-CoA carboxylase, stearoyl-CoA desaturase 1 (SCD1), and ATPcitrate lyase (ACL). Biochemical analysis showed that protein levels andactivities of the lipogenic enzymes were reduced in liver extracts offatostatin-treated mice (FIGS. 25A-25C). Thus, fatostatin blocks theprocessing of SREBP-1 in liver, downregulates lipogenic enzymes, andreduces hepatic triglyceride storage. Fatostatin represents the firstnon-sterol-like synthetic molecule that inhibits the activation ofSREBPs.

Luciferase reporter assay. On day 0, CHO-K1 cells were plated out onto a96-well plate in medium A (a 1:1 mixture of Ham's F-12 medium andDulbecco's modified Eagle's medium, with 5% fetal bovine serum, 100units/mL penicillin, and 100 μg/mL streptomycin sulfate). On day 2, thecells were transiently co-transfected with pSRE-Luc (an SRE-1-drivenluciferase reporter construct) (Hua et al., 1995) and pAc-β-gal (β-galreporter in which the expression of β-gal is controlled by an actinpromoter), using Lipofectamine reagent (Invitrogen). After incubationfor 5 h, the cells were washed with phosphate-buffered saline (PBS), andthen incubated, in the absence or presence of fatostatin, in medium B (a1:1 mixture of Ham's F-12 medium and Dulbecco's modified Eagle's medium,with 5% lipid-depleted serum, 100 units/mL penicillin, 100 μg/mLstreptomycin sulfate, 50 mM compactin, and 50 mM sodium mevalonate).After 20 h of incubation, the cells in each well were lysed, andaliquots were used to measure luciferase and β-galactosidase activities.Luciferase activity was normalized by the activity of β-galactosidase.For overexpression of the N-terminal matured form of SREBP-1c,pCMV-SREBP-1c(1-436) was co-transfected with pSRE-Luc and pAc-β-gal.

Western blot analysis of SREBP processing. On day 0, CHO-K1 cells wereplated out onto a 100 mm dish of medium A. On day 2, the cells werewashed with PBS, and then incubated in medium B in the absence orpresence of fatostatin. On day 3, the cells were washed once with coldPBS, and then treated with buffer containing 10 mM Tris-HCl, pH 7.6, 100mM NaCl, 1% (w/v) SDS, and protease inhibitor mixture (1 μg/ml pepstatinA, 10 μg/ml leupeptin, 200 μM phenylmethylsulfonyl fluoride). Theprotein concentration of each total cell extract was measured (BCA kit;Pierce), after which a 22-33 μg aliquot of cell extract was mixed with0.25 volume of buffer (250 mM Tris-HCl, pH 6.8, 10% SDS, 25% glycerol,0.2% (w/v) bromophenol blue, and 5% (v/v) 2-mercaptoethanol), heated for7 min at 95° C. The samples were separated on a 10% SDS-PAGE gel andblotted using mouse monoclonal antibody against SREBP-2 (IgG-7D4) (Yanget al., 1995). The specific bands were visualized using enhancedchemiluminescent (ECL) detection reagents (Amersham).

Modification of SCAP oligosaccharides. Cell membrane fractions wereprepared as described elsewhere herein. The membrane pellets wereresuspended in 0.1 mL of buffer containing 10 mM Hepes.KOH (pH 7.4), 10mM KCl, 1.5 mM MgCl₂, 1 mM sodium EDTA, and 100 mM NaCl. Aliquots ofprotein were then incubated in the absence or presence of 1 μg oftrypsin, in a total volume of 58 μL, for 30 min at 30° C. Reactions werestopped by addition of 2 μL (400 units) of soybean trypsin inhibitor.For subsequent treatment with endoglycosidase H, individual samplesreceived 10 μl of solution containing 3.5% (wt/vol) SDS and 7% (vol/vol)2-mercaptoethanol. After heating at 100° C. for 10 min, each samplereceived sequential additions of 9 μl of 0.5 M sodium citrate (pH 5.5),5 μL of solution containing 17′ protease inhibitors (a concentration of1×, corresponding to 10 μg/mL leupeptin, 5 μg/mL pepstatin A, and 2μg/mL aprotinin), followed by 1 μL (5 units) of endoglycosidase H. Thereactions were carried out overnight at 37° C. and stopped by theaddition of 20 μL of buffer containing 0.25 M Tris-HCl (pH 6.8), 2% SDS,10% (vol/vol) glycerol, 0.05% (wt/vol) bromophenol blue, and 4%2-mercaptoethanol. The mixtures then were heated at 100° C. for 5 minand subjected to SDS/PAGE (12% gels).

Confocal microscopic analyses. CHO-K1 cells on a glass-bottom 96-wellplate (Grainer) at ˜70% confluency were incubated with 0.2 μM ER-trackerRed (Invitrogen) and 5 mM dansyl fatostatin for 1 h. Fluorescent imageswere captured and analyzed with a Carl Zeiss LSM 510 confocalmicroscope, equipped with a CSU10 spinning-disk confocal scanner(Yokogawa Electric Corporation) and an ORCA-CCD camera (HamamatsuPhotonics). Images were analysed with IPLab software (Solution Systems).

Binding assay. Cell membrane fractions were prepared as described inSupplementary Methods. The membrane fraction was extracted with PBScontaining 0.1% FOS-Choline 10 (Hampton Research). The extract was mixedwith Neutravidine-agarose beads (10 μL) saturated with biotinylatedfatostatin and incubated for 1 h. The bound proteins were washed fourtimes with PBS containing 0.1% FOS-Choline 10, boiled in 25 μL of SDSsample buffer, and subjected to western blotting. For the competitionassay, saturated amounts of cholesterol or fatostatin were added to themembrane extract before incubating with the beads.

Animal studies procedures. Four to five week-old homozygous male obese(ob/ob) mice (C57BL/6J, The Jackson Laboratory, Bar Harbor, Me.) werehoused under controlled conditions (12 h light/dark cycle; 25° C.). Theanimals were housed 5 per cage, and had ad libitum access to standardlaboratory chow (Purina Mills, Richmond, Ind.) and water for one weekafter their arrival. On first day of the experiment and every daythereafter, the weight of each mouse and the amount of food intake weremeasured between 3:00 and 5:00 p.m. Following weight measurements,treated mice received an ip injection of fatostatin (30 mg/kg; 150 μL),and control mice received 10% DMSO in PBS. Daily Injections werecontinued for four weeks, until the end of the study.

Blood constituents. After 28 days of daily injection of fatostatin micewere fasted for 5-6 h, whole blood glucose and 3-hydroxybutyrate weremeasured with a Glucometer Precision Xtra (Abbott). Measurements of theserum constituents, glucose, triglyceride, and cholesterol, wereperformed by the Comparative Pathology Laboratory (Baylor College ofMedicine). Serum non-esterified fatty acids (NEFA) were measured using aNEFA C kit (Wako Chemicals, Richmond, Va.).

Liver analyses. Mice were sacrificed, and weights of livers andepididymal fat pads were determined. Frozen sections of liver slicesfrom individual animals were stained with Oil Red 0 to visualize the fatdroplets (triglycerides) in liver slices, as described (Abu-Elheiga etal., 2001). The remaining liver tissues were frozen in liquid nitrogenand kept at −80° C. for further analysis.

Tissue triglyceride and cholesterol contents. Liver triglyceride andcholesterol contents were determined as described by Chandler et al.(2003), using a Cholesterol E Kit (Wako) and an Infinity TriglycerideKit (Thermo Electron, Melbourne, Australia).

Synthesis of Fatostatin 1, Dansyl Fatostatin and Fatostatin-PolyprolineLinker-Biotin Conjugate

FIG. 23 depicts the synthesis of fatostatin 1, dansyl fatostatin,fatostatin-polyproline linker-biotin conjugates and the syntheticintermediates.

Synthesis of Fatostatin 1

A mixture of prothionamide (1.03 g, 5.70 mmol) and2-bromo-4′-methylacetophenone (1.22 g, 5.70 mmol) in ethanol (20 ml) washeated at 70° C. with stirring for 0.5 h, and then cooled to 0° C. Ayellow precipitate formed was filtered, washed with cold ethanol, anddried to give 2-propyl-4-(4-p-tolylthiazol-2-yl)pyridine (fatostatin) 1HBr salt (1.78 g, 83%) as yellow needles. mp: 190-193° C.; ¹H NMR (600MHz, DMSO-d₆): δ 8.88 (d, J=6.2 Hz, 1H), 8.54 (s, 1H), 8.46 (d, J=1.4Hz, 1H), 8.36 (dd, J=1.4, 6.2 Hz, 1H), 7.99 (d, J=7.6 Hz, 2H), 7.31 (d,J=7.6 Hz, 2H), 3.03 (t, J=7.6 Hz, 2H), 2.35 (s, 3H), 1.80 (m, 2H), 0.96(t, J=7.6 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆): δ 161.3, 158.5, 156.9,146.2, 143.2, 138.4, 130.4, 129.5, 126.3, 122.3, 120.3, 119.5, 35.0,22.4, 20.9, 13.4; HRMS (m/z): [M+H]⁺ calcd for C₁₈H₁₉N₂S, 295.1269.found, 295.1269.

Synthesis of 4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)benzenamine 16

A pressure tube was charged with 2 (1.08 g, 3.0 mmol), benzophenoneimine (0.57 g, 3.3 mmol), Pd₂(dba)₃ (86 mg, 0.15 mmol), BINAP (280 mg,0.45 mmol), sodium tert-butoxide (1.44 g, 9.0 mmol), and dry toluene (30mL) and purged with argon gas. The pressure tube was sealed and heatedin a 100° C. bath for 20 h. After being cooled to room temperature, thereaction mixture was chromatographed (SiO₂, 4:1 hexane:EtOAc) to provide1.35 g of 8 (98%) as a yellow oil. Then to a solution of 8 (1.35 g, 2.9mmol) in THF (20 mL) was added 2 N aqueous HCl solution (15 mL). Afterbeing stirred at room temperature for 2 h, the reaction mixture wasconcentrated under reduced pressure, then diluted with EtOAc (100 mL)and washed with saturated Na₂CO₃ (50 mL) solution. The aqueous wash wasextracted with EtOAc (3×40 mL), and the combined EtOAc layers were driedover Na₂SO₄ and concentrated. The crude product was chromatographed(SiO₂, 4:1 hexane: EtOAc) to provide 0.73 g of4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)benzenamine 16 (82%) as a whitecrystal. ¹H NMR (600 MHz, CDCl₃): δ 8.61 (d, J=4.8 Hz, 1H), 7.80 (d,J=8.9 Hz, 2H), 7.75 (d, J=1.4 Hz, 1H), 7.67 (dd, J=1.4, 4.8 Hz, 1H),7.36 (s, 1H), 6.75 (d, J=8.9 Hz, 2H), 3.82 (brs, 1H), 2.85 (t, J=7.6 Hz,2H), 1.83 (m, 2H), 1.01 (t, J=7.6 Hz, 3H); ¹³C NMR (150 MHz, CDCl₃): δ164.9, 163.4, 157.3, 150.0, 146.8, 140.8, 127.7, 124.8, 119.2, 117.8,115.1, 111.3, 40.4, 23.1, 13.9; HRMS (m/z): [M+H]⁺ calcd for C₁₇H₁₈N₃S,296.1221. found, 296.1228.

Synthesis of Dansyl Fatostatin

To a magnetically stirred solution of 16 (50 mg, 0.17 mmol) and pyridine(27 mg, 0.34 mmol) in CH₂Cl₂ (5 mL) was added dansyl chloride (50 mg,0.18 mmol). After being stirred for 17 h, the reaction mixture wasconcentrated under reduced pressure, and the residue was partitionedbetween EtOAc (50 mL) and saturated NH4Cl solution (20 mL). The aqueousphase was extracted with EtOAc (2×20 mL). The combined extracts werewashed with saturated NaHCO₃ solution, dried over Na₂SO₄, andconcentrated. The crude product was chromatographed (SiO₂, 2:1 hexane:EtOAc) to afford dansylfatostatin (65 mg, 73%) as a yellow crystal. ¹HNMR (600 MHz, CDCl₃): δ 8.60 (d, J=4.8 Hz, 1H), 8.50 (d, J=8.2 Hz, 1H),8.36 (d, J=8.3 Hz, 1H), 8.21 (d, J=8.3 Hz, 1H), 7.76 (d, J=8.6 Hz, 2H),7.70 (d, J=1.5 Hz, 1H), 7.62 (dd, J=1.5, 4.8 Hz, 1H), 7.61 (t, J=8.3 Hz,1H) 7.44 (s, 1H), 7.43 (dd, J=7.5, 8.2 Hz, 1H), 7.19 (d, J=7.5 Hz, 1H),7.04 (d, J=8.6 Hz, 2H), 6.97 (brs, 1H), 2.87 (s, 6H), 2.84 (t, J=7.6 Hz,2H), 1.81 (m, 2H), 1.00 (t, J=7.6 Hz, 3H); ¹³C NMR (150 MHz, CDCl₃): δ165.5, 163.5, 156.1, 152.2, 150.0, 140.5, 136.7, 134.0, 131.1, 131.0,130.5, 129.8, 129.7, 128.7, 127.3, 123.1, 121.6, 119.2, 118.3, 117.8,115.3, 113.8, 45.4, 40.4, 23.1, 13.9; HRMS (m/z): [M+H]⁺ calcd forC₂₉H₂₉N₄O₂S₂, 529.1732. found, 529.1733.

Synthesis of tert-butyl4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenylcarbamate 40

To a magnetically stirred solution of 16 (0.57 g, 1.92 mmol) and4-(dimethylamino)pyridine (5 mg, 0.4 mmol) in THF (20 mL) was addeddi(tert-butyl)dicarbonate (0.49 g, 2.21 mmol). After being stirred for17 h, the reaction mixture was concentrated under reduced pressure, andthe residue was partitioned between EtOAc (100 mL) and saturated NH₄Clsolution (30 mL). The aqueous phase was extracted with EtOAc (2×50 mL).The combined extracts were washed with saturated NaHCO₃ solution, driedover Na₂SO₄, and concentrated. The crude product was chromatographed(SiO₂, 2:1 hexane: EtOAc) to afford tert-butyl4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenylcarbamate 40 (0.33 g, 43%)as a yellow foam. ¹H NMR (300 MHz, CDCl₃): δ 8.68 (d, J=5.1 Hz, 1H),7.93 (d, J=8.4 Hz, 2H), 7.79 (s, 1H), 7.72 (d, J=5.1 Hz, 1H), 7.52 (s,1H), 7.47 (d, J=8.4 Hz, 2H), 6.58 (s, 1H), 2.90 (t, J=7.5 Hz, 2H,), 1.87(m, 2H), 1.55 (s, 9H), 1.03 (t, J=7.5 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃):δ 163.1, 156.5, 152.3, 149.6, 140.6, 138.5, 128.7, 128.1, 127.0, 118.3,117.7, 114.3, 112.9, 80.6, 41.0, 28.4, 24.1, 13.7; HRMS (m/z): [M+H]⁺calcd for C₂₂H₂₆N₃O₂S, 396.1746. found, 396.1738.

Synthesis of R═N(Boc)CH₂CH₂CH₂COOH intermediate

In an N₂ atmosphere, 40 (200 mg, 0.51 mmol) was added to a suspension ofNaH (60% dispersion in mineral oil, 24 mg, 0.6 mmol) in DMF (5 mL) andthe mixture was stirred for 2 hr at room temperature. Then NaI (91 mg,0.6 mmol) and ethyl 4-bromobutyrate (0.12 g, 0.6 mmol) in DMF (2 mL)were added. After being stirred for 18 h, the reaction mixture waspoured into water (20 mL) and was extracted with EtOAc (2×50 mL). Thecombined extracts were dried over Na₂SO₄, and concentrated. The crudeproduct was chromatographed (SiO₂, 2:1 hexane:AcOEt) to provide 11 (35mg, 13%) as a yellow oil. Then to a solution of 11 (30 mg, 2.9 mmol) inTHF (1 mL) and MeOH (0.5 mL) was added 2 N aqueous NaOH solution (0.2mL). After being stirred at room temperature for 18 h, the reactionmixture was concentrated under reduced pressure, then diluted with EtOAc(10 mL) and washed with saturated NH₄Cl solution (5 mL). The aqueouswash was extracted with EtOAc (2×10 mL), and the combined EtOAc layerswere dried over Na₂SO₄ and concentrated. The crude product waschromatographed (SiO₂, EtOAc) to provide 14 mg of theR═N(Boc)CH₂CH₂CH₂COOH intermediate (50%) as a white foam. ¹H NMR (300MHz, CDCl₃): δ 8.69 (d, J=5.1 Hz, 1H), 7.95 (d, J=8.4 Hz, 2H), 7.80 (s,1H), 7.73 (d, J=5.1 Hz, 1H), 7.53 (s, 1H), 7.51 (d, J=8.4 Hz, 2H), 6.58(s, 1H), 2.92 (m, 4H), 2.33 (m, 2H), 1.90 (m, 4H), 1.50 (s, 9H), 1.02(t, J=7.5 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 174.2, 162.9, 156.3,152.9, 149.6, 140.6, 138.5, 129.3, 128.9, 127.2, 119.0, 117.9, 114.8,113.3, 80.8, 41.0, 40.3, 32.1, 28.4, 24.1, 21.1, 13.7; HRMS (m/z):[M+H]⁺ calcd for C₂₆H₃₂N₃O₄S, 482.2114. found, 482.2120.

Synthesis of4-(4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenylamino)-N-isopropylbutanamide

To a solution of the R═N(Boc)CH₂CH₂CH₂COOH intermediate (17 mg, 0.035mmol), triethylamine (17 μl, 0.14 mmol) and isopropylamine (4 μL, 0.046mmol) in DMF (0.5 mL) was HATU (16 mg, 0.042 mmol). After being stirredfor 18 h, the reaction mixture was concentrated under reduced pressure,and the residue was partitioned between EtOAc (20 mL) and saturatedNH₄Cl solution (10 mL). The aqueous phase was extracted with EtOAc (2×10mL). The combined extracts were washed with saturated NaHCO₃ solution,dried over Na₂SO₄, and concentrated. The crude product waschromatographed (SiO₂, 2:1 hexane: EtOAc) to afford the N-Boc protected4-(4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenylamino)-N-isopropylbutanamide (14 mg, 77%) as a yellow oil. Then to a solution of 13 (12mg, 2.9 mmol) in THF (1 mL) was added TFA (0.2 mL). After being stirredat room temperature for 18 h, the reaction mixture was concentratedunder reduced pressure, then diluted with EtOAc (20 mL) and washed withsaturated NH₄Cl solution (10 mL). The aqueous wash was extracted withEtOAc (2×5 mL), and the combined EtOAc layers were dried over Na₂SO₄ andconcentrated. The crude product was chromatographed (SiO₂, 2:1 hexane:EtOAc) to provide 6.2 mg of4-(4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenylamino)-N-isopropylbutanamide (63%) as a yellow foam. ¹H NMR (300 MHz, CDCl₃): δ 8.69 (d,J=5.4 Hz, 1H), 8.04 (1H, s), 7.96 (d, J=8.7 Hz, 2H), 7.96 (s, 1H), 7.72(d, J=5.4 Hz, 1H), 7.51 (s, 1H), 6.65 (d, J=8.7 Hz, 2H), 3.95 (m, 1H),3.03 (m, 4H,), 2.35 (m, 2H), 1.92 (m, 4H), 1.25 (d, J=6.3 Hz, 6H), 1.03(t, J=7.5 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 171.6, 162.9, 156.3,152.9, 140.6, 138.5, 129.3, 128.9, 127.2, 119.0, 117.9, 114.8, 113.3,42.2, 41.0, 40.3, 32.1, 24.1, 23.2, 21.1, 13.7; HRMS (m/z): [M+H]⁺ calcdfor C₂₄H₃₁N₄OS, 423.2219. found, 423.2216.

Synthesis of Fatostatin-Polyproline Linker-Biotin Conjugate

Fatostatin-KPGQFLYELKKPPPPPPPPPKK (SEQ ID NO: 15)-aminocaproicacid-biotin.

Conjugates were synthesized on Rink-Amide MBHA resin by couplingN-α-Fmoc-protected amino acids, N-ε-Fmoc-e-aminocaproic acid,R═N(Boc)CH₂CH₂CH₂COOH intermediate, biotin, purified by a reversed phaseHPLC as described (Sato et al., 2007). Conjugate 3: calcd forC₁₅₅H₂₃₈N₃₅O₂₉S₂ ⁺ requires 3119.9. Found (MALDI-TOF-MS) 3119.7 [M+H]⁺.

Plasmids. pSRE-Luc, pCMV-SREBP-1c(1-436), pCMV-PLAP-BP2(513-1141) andpCMV-SCAP were gifts from J. L. Goldstein and M. S. Brown (University ofTexas Southwestern Medical Center) (Sakai et al., 1998; Hua et al.,1995).

Antibodies. Monoclonal anti-SREBP-1 IgG (2A4), anti-SCAP IgG (9D5) andanti-ATF6 IgG (H-280) were purchased from Santa Cruz Biotechnology.Monoclonal anti-SREBP-2 IgG-7D4 was a gift from J. L. Goldstein and M.S. Brown (University of Texas Southwestern Medical Center). Polyclonalanti-FAS, anti-ACC, anti-SCD1 and anti-ACL IgG were purchased from BDBiosciences.

Cell culture. Chinese hamster ovary cells K1 (CHO-K1) cells weremaintained in a Dulbecco's modified Eagle's medium/Ham's F12 medium[1:1] with 5% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mLstreptomycin sulfate at 37° C. under 5% CO₂. Human androgen-independentprostate cancer cells (DU145) were maintained in an Eagle's minimumessential medium containing 2 mM L-glutamine, 1.0 mM sodium pyruvate,0.1 mM nonessential amino acids, and 1.5 g/L sodium biocarbonate with10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mLstreptomycin sulfate at 37° C. under 5% CO₂.

Preparation of cell membrane fraction. Cells were harvested, and thenresuspended in buffer (10 mM Hepes.KOH (pH 7.4), 10 mM KCl, 1.5 mM MgCl₂and 1 mM sodium EDTA), passed through a 22-gauge needle, and centrifugedat 1,000×g for 5 min. The postnuclear supernatants then were centrifugedat 15,000×g for 30 min, and the supernatant was removed.

Oligonucleotide microarray analysis. DU145 prostate cancer cells weretreated with 5 mM of fatostatin or DMSO alone in the presence of 1 μg/mLof IGF1 for 6 hrs in a serum free medium, total RNA was extracted in aTRI reagent (Molecular Research Center) and further isolated by RNeasyMini Kit (Qiagen). Purified mRNA was analyzed in Baylor College ofMedicine Microarray Core Facility by Affymetrix Human Genome U133 Plus2.0 Array consisting of almost 45,000 probe sets representing more than39,000 transcripts derived from approximately 33,000 well-substantiatedhuman genes (Affymetrix, Inc.).

RT-PCR experiments. Total RNA was extracted from DU145 cells in TRIreagent (Molecular Research Center) and isolated with an RNeasy Mini Kit(Qiagen). The RNA sample was subjected to RT-PCR by using the AccessRT-PCR System (Promega). RT-PCR reactions contained total RNA, 1 μM ofeach primer, 0.2 mM dNTP, 1 mM MgSO₄, AMV reverse transcriptase (2units), and Tfl DNA polymerase (2 units) in a final volume of 25 μL. Theprimer pairs used are as follows: 5′-TCA GAC CGG GAC TGC TTG GAC GGC TCAGTC-3′ (SEQ ID NO: 16) and 5′-CCA CTT AGG CAG TGG AAC TCG AAG GCC G-3′(SEQ ID NO: 17) for Low density lipoprotein receptor (LDLR); 5′-GCC TGCTTG ATA ATA TAT AAA C-3′ (SEQ ID NO: 18) and 5′-CAC TTG AAT TGA GCT TTAG-3′ (SEQ ID NO: 19) for stearoyl-CoA desaturase (SCD); 5′-AAG AAA AAGTGT CAG ACA GCT GG-3′ (SEQ ID NO: 20) and 5′-TGG ACT GAA GGG GTG TTAGC-3′ (SEQ ID NO: 21) for ATP citrate lyase (ACL); 5′-GCC CGA CAG TTCTGA ACT GGA ACA-3′ (SEQ ID NO: 22) and 5′-GAA CCT GAG ACC TCT CTG AAAGAG-3′ (SEQ ID NO: 23) for 3-hydroxy-3-methylglutaryl-CoA reductase(HMG-CoAR); 5′-CTG CCT GAC TGC CTC AGC-3′ (SEQ ID NO: 24) and 5′-ACC TCTCCT GAC ACC TGG G-3′ (SEQ ID NO: 25) for mevalonate pyrophosphatedecarboxylase (MVD); 5′-AAG ACT TCA GGG TAA GTC ATC A-3′ (SEQ ID NO: 26)and 5′-CGT GTA TAA TGG TGT CTA TCA G-3′ (SEQ ID NO: 27) for insulininduced gene 1 (INSIG1). The amplification conditions are as follows: 1cycle at 94° C. for 4 min, then denatured at 94° C. for 40 s, annealedat 50° C. for 40 s, and extended at 68° C. for 2 min with 22 cycles forSCD and HMG CoA R, annealed at 58° C. with 24 cycles for LDLR andINSIG1, or annealed at 60° C. with 24 cycles for ACL, annealed at 55° C.with 30 cycles for MVD. The amplified DNAs were analyzed by an agarosegel and quantified with the Scion-image software.

PLAP-BP2 Cleavage. On day 0, CHO-K1 cells were plated out onto a 96-wellplate in medium A. On day 2, the cells were transiently co-transfectedwith pCMV-PLAP-BP2(513-1141), pCMV-SCAP and pAc-β-gal, usingLipofectamine reagent (Invitrogen). After incubation for 5 h, the cellswere washed with PBS, and then incubated, in the absence or presence offatostatin (20 μM) or sterols (10 μg/mL cholesterol and 1 μg/mL25-hydroxycholesterol), in medium B. After 20 h of incubation, analiquot of the medium was assayed for secreted alkaline phosphataseactivity. The cells in each well were lysed, and used for measurement ofβ-galactosidase activities. The alkaline phosphatase activity wasnormalized by the activity of β-galactosidase.

Enzymatic activities and western blot analyses. A portion of the frozenliver was ground to powder in liquid nitrogen. The powdered tissues weresuspended in 10 mL of PBS containing 0.1 mM PMSF, 5 mM benzamidine, and5 mg/mL protease inhibitor cocktail (Roche), and homogenized usingPolytron (3×30 Sec, at high speed), and sonicated briefly to degradeDNA. The extracts were clarified by centrifugation at 16,000×g for 20min. Protein concentrations in the supernatant were determined, andsubjected to western blot analysis using antibodies against FAS, ACC,SCD1 and ACL. The intensity of the specific bands of proteins ofinterest were scanned and normalized against b-actin forquantifications. FAS and ACC activities from the liver extracts weredetermined as described earlier (Mao et al., 2006).

Example 11 Synthesis ofbenzyl(R)-2-(4-(4-(methylsulfonamido)phenyl)thiazol-2-yl)pyrrolidine-1-carboxylate

As shown in FIG. 26, to a solution of L-prolinamide (1.14 g, 10.0 mmol)in acetonitrile (100 mL) were added benzyl chloroformate (1.55 mL, 11.0mmol) and triethylamine (2.79 mL, 20 mmol) at rt. The mixture wasstirred at rt for 12 h and diluted with AcOEt. The mixture was pouredinto 0.1 M HCl solution and separated between the organic layer and theaqueous layer. The organic layer was washed with sat. aq. NaHCO₃ andbrine, dried (Na₂SO₄), filtered, and concentrated. The residue waspurified by silica gel column chromatography to give 67 (2.26 g, 91%) ascolorless oil.

A mixture of 67 (586 mg, 2.36 mmol) and Lawesson's reagent (1.05 g, 2.60mmol) in toluene (30 mL) was stirred at 90° C. for 1 h. After cooling tort, the solvent was evaporated. The residue was purified by silica gelcolumn chromatography to give 68 (495 mg, 79%) as a white amorphoussolid.

A mixture of 68 (300 mg, 1.13 mmol) and 4-bromophenacyl bromide (318 mg,1.13 mmol) in EtOH (10 mL) was stirred under reflux for 2 h. Afterevaporated, the residue was purified by silica gel column chromatographyto give 48 (265 mg, 53%) as a white solid.

A mixture of 48 (230 mg, 0.519 mmol), Pd₂(dba)₃ (9.50 mg, 2 mol %),BINAP (97%, 20.0 mg, 6 mol %), sodium t-butoxide (69.8 mg, 0.727 mmol),and benzophenone imine (0.105 mL, 0.623 mmol) in toluene (5.0 mL) underreflux for 8 h. After cooling to rt, the mixture was filtered throughCelite and the filtrate was evaporated. The residue was purified bysilica gel column chromatography to give 69 (228 mg, 81%) as a yellowamorphous solid.

To a solution of 69 (175 mg, 0.321 mmol) in THF (3 mL) was added aqueousHCl solution (4 M, 0.3 mL) and the mixture was stirred at rt for 1 h.The mixture was neutralized with sat. aq. NaHCO₃ and extracted withAcOEt. The organic layer was washed with brine, dired (Na₂SO₄),filtered, and concentrated. The residue was purified by silica gelcolumn chromatography to give 70 (113 mg, 93%) as a colorless oil.

A mixture of 70 (92 mg, 0.24 mmol), methanesulfonyl chloride (0.021 mL,0.27 mmol), and pyridine (0.043 mL, 0.53 mmol) in CH₂Cl₂ (2 mL) wasstirred at rt for 8 h. After addition of MeOH, the mixture was dilutedwith AcOEt and washed with sat. aq. NaHCO₃ and brine. The organic layerwas dried (Na₂SO₄), filtered, and concentrated. The residue was purifiedby silica gel column chromatography to give 53 (91 mg, 83%) as a whitesolid.

¹H NMR (CDCl₃, 600 MHz, two rotamers, ratio=1:0.95) δ 7.80-7.86 (m, 2H),7.14-7.41 (m, 7H), 6.64 (s, 1H, major rotamer), 6.83 (s, 1H, minorrotamer), 5.07-5.36 (m, 3H), 3.73 (m, 1H), 3.59 (m, 1H), 3.03 (s, 3H,major rotamer), 3.00 (s, 3H, minor rotamer), 2.28-2.39 (m, 2H),2.00-2.10 (m, 2H); ¹³C NMR (CDCl₃, 150 MHz, two romamers, major peaksare shown) 174.7, 155.4, 154.4, 136.4, 131.9, 128.5, 128.3, 127.9,127.8, 127.7, 127.7, 120.8, 112.3, 67.1, 59.4, 47.2, 39.4, 32.9, 23.1;HRMS (FAB): Exact mass calcd. for C₂₂H₂₄N₃O₄S₂ [M+H]⁺, 458.1203. Found458.1200.

Example 12 Synthesis ofN-(4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenyl)methanesulfonamide(19)

As shown in FIG. 27, in reaction (a) a mixture of prothionamide (9.01 g,50.0 mmol) and 4-bromophenacyl bromide 6a (13.9 g, 50.0 mmol) in EtOH(400 mL) was stirred at 70° C. for 1 h. After cooled to roomtemperature, the precipitation was filtered off with EtOAc and washedwith EtOAc and saturated aqueous NaHCO₃. The organic layer was washedwith brine, dried over Na₂SO₄, and concentrated to give4-(4-bromophenyl)-2-(2-propylpyridin-4-yl)thiazole 2 (14.3 g, 79%) as alight-yellow solid.

In reaction (b) a pressure tube was charged with 2 (1.08 g, 3.0 mmol),benzophenone imine (0.57 g, 3.3 mmol), Pd₂dba₃ (86 mg, 0.15 mmol), BINAP(280 mg, 0.45 mmol), sodium tert-butoxide (1.44 g, 9.0 mmol), and drytoluene (30 mL) and purged with argon gas. The pressure tube was sealedand heated in a 100° C. bath for 20 hrs. After being cooled to roomtemperature, the reaction mixture was chromatographed (SiO₂, 4:1hexane:EtOAc) to provide 1.35 g of 71 (98%) as a yellow oil.

In reaction (c) then to a solution of 71 (1.35 g, 2.9 mmol) in THF (20mL) was added 2 M aqueous HCl solution (15 mL). After being stirred atroom temperature for 2 hrs, the reaction mixture was concentrated underreduced pressure, then diluted with EtOAc (100 mL) and washed withsaturated Na₂CO₃ (50 mL) solution. The aqueous wash was extracted withEtOAc (3×40 mL), and the combined EtOAc layers were dried over Na₂SO₄and concentrated. The crude product was chromatographed (SiO₂, 4:1hexane:EtOAc) to provide 0.73 g of4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)benzenamine 16 (82%) as a whitecrystal.

¹H NMR (600 MHz, CDCl₃): δ 8.61 (d, J=4.8 Hz, 1H), 7.80 (d, J=8.9 Hz,2H), 7.75 (d, J=1.4 Hz, 1H), 7.67 (dd, J=1.4, 4.8 Hz, 1H), 7.36 (s, 1H),6.75 (d, J=8.9 Hz, 2H), 3.82 (brs, 1H), 2.85 (t, J=7.6 Hz, 2H), 1.83 (m,2H), 1.01 (t, J=7.6 Hz, 3H); ¹³C NMR (150 MHz, CDCl₃): δ 164.9, 163.4,157.3, 150.0, 146.8, 140.8, 127.7, 124.8, 119.2, 117.8, 115.1, 111.3,40.4, 23.1, 13.9; HRMS (m/z): [M+H]+ calcd for C17H18N3S, 296.1221.found, 296.1228.

In reaction (d) methanesulfonyl chloride (0.23 mL, 2.97 mmol) was addedto a stirred solution of 16 (800 mg, 2.71 mmol) and pyridine (0.66 mL,8.1 mmol) in CH₂Cl₂ (20 mL) at 0° C. After being stirred for 0.5 h, themixture was poured into 2 M citric acid solution and extracted withEtOAc. The combined extracts were washed with saturated NaHCO₃ solutionand brine, dried over Na₂SO₄, and concentrated to produceN-(4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenyl)methanesulfonamide 19(880 mg, 87%) as a yellow foam.

¹H NMR (300 MHz, CD₃OD): δ 8.55 (d, J=5.2 Hz, 1H), 8.02 (d, J=8.8 Hz,2H), 7.95 (s, 1H), 7.90 (d, J=1.9 Hz, 1H), 7.84 (dd, J=1.9, 5.2 Hz, 1H),7.34 (d, J=8.8 Hz, 2H), 3.00 (s, 3H), 2.86 (t, J=7.7 Hz, 2H), 1.80 (m,2H), 1.01 (t, J=7.3 Hz, 3H). m/z=374 [M+H]⁺.

Example 13 Synthesis ofN-isopropyl-4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)benzamine (17)

As shown in FIG. 27, the procedure to synthesize compound 17 is utilizesthe same steps a-c as described for compound 19 in Example 12. Forcompound 17 at step e, acetone (2.5 mL, 34.5 mmol) and acetic acid (2.0mL, 34.5 mmol) were added to a stirred solution of 16 (1.02 g, 3.45mmol) in CH₂Cl₂ (20 mL). After the mixture was stirred for 1 h,Na(OAc)₃BH (1.5 g, 6.9 mmol) was added. The mixture was stirred for 20h. The mixture was poured into saturated NaHCO₃ solution and extractedwith EtOAc. The combined extracts were dried over Na₂SO₄ andconcentrated. Chromatography of the crude product (SiO₂, 4:1hexane/EtOAc) produced 17 (845 mg, 73%) as a white foam. ¹H NMR (300MHz, CDCl₃): δ 8.61 (d, J=5.2 Hz, 1H), 7.81 (d, J=8.5 Hz, 2H), 7.76 (d,J=1.4 Hz, 1H), 7.68 (dd, J=1.4, 5.2 Hz, 1H), 7.34 (s, 1H), 6.65 (d,J=8.5 Hz, 2H), 3.70 (m, 1H), 2.86 (t, J=7.6 Hz, 2H), 1.83 (m, 2H), 1.25(d, J=6.0 Hz, 6H), 1.02 (t, J=7.4 Hz, 3H). m/z=338 [M+H]⁺.

Example 14 SREBP Activation Assays for Compounds 45-66

Standard SREBP activation assays were performed on the exemplarycompounds identified in Table 4 as per the method for Fatostatin A inExample 1. The ability of endogenous SREBPs to activate transcription ofan SREBP-responsive reporter gene was measured in the presence orabsence of compounds 45-55 and 19 (FIG. 28), compounds 56-61 and 19(FIG. 29) and compounds 62-66 (FIG. 30) at 5 μM in CHOK1 cells. Ofthese, compound 61 demonstrated inhibition of SREBP activation at about25%, with compound 53 at about 30%, compound 58 at about 42%, andcompound 19 at about 45%. The inhibitory concentrations of compounds 53(FIG. 31) and compounds 58 and 61 (FIG. 32) were determined.

Example 15 Compound 19 Inhibited the Growth of the Human Breast CancerCell Lines SUM159 and Downregulated Lipogenic Pathways

Treating SUM159 cells with different concentrations of compound 19 for48 hours caused an inhibition of cell growth and a toxic effect (FIG.33A). Cells seeded onto 96 well plates at a density of 5000 cells/wellin 100 μl medium containing 2% charcoal treated serum. After 24 hoursdifferent concentrations of compound 19 were added for an additional 48hours. Cell viability was measured using WST-1 Cell Viability andProliferation Assay (ScienCell Research Laboratory, Carlsbad Calif.).Changes in expression levels of lipogenic genes mRNA was determined incells that were treated with 10 μM. There was a significantdownregulation of the genes that are under the control of transcriptionfactors, SREBPs. Insig2 gene which is not known to be regulated by thesetranscription factors was not affected by the treatment (FIG. 33B).

The effect of Compound 19 was also demonstrated on human hepatocellularcarcinoma cell line (HePG2). As shown in FIGS. 34A-34B, Compound 19produced a high level of toxicity as evidenced by the dramatic change inthe morphology and the inhibition of growth of the treated cells. WhenHePG2 cells were treated with 25 μM of Compound 19 there was asignificant decrease in the level of the mature and active form and anincrease in the precursor of SREBP-1 and consequently the expressionlevels of genes that are controlled by SREBP (FIGS. 34A-34B). Theseresults are consistent and supportive of the conceptual action ofcompound 19 as an inhibitor of lipogenesis through the inhibition ofSREBP activation.

Compound 19 Inhibits the Growth of Human Acute Lymphoblastic LeukemiaCell Line MOLT-4

MOLT-4 cells were grown at two different conditions under 5% fetalbovine serum (FBS) and under 5% FBS fat free serum (charcoal treatedserum). 10,000 Cells were seeded a 96 well plate for 24 hours beforestarting the treatment with different concentrations of compound 19.Compound 19 dissolved in DMSO was added to cells for 48 hours beforedetermining cell growth using MTT assay. As shown in FIG. 35A, thegrowth of the cells under 5% FBS and FBS fat free was similar. Compound19 inhibited cell growth about 75-80% at 10 and 20 μM under both growthconditions. These results show that at 10 and 20 μM compound 19 wasequally effective even in the presence of exogenous lipids, whichsuggest that inhibition of de novo lipid synthesis result in growthinhibition in this cancer sell line. Interestingly, under FBS fat freeserum there was 30-50% growth inhibition at 2 and 5 μM compound 19 (FIG.35C) and a strong trend of inhibition at 5 μM in 5% FBS condition (FIG.35B).

Compound 19 Inhibits the Growth of Human Multiple Myeloma Cell LineRPMI8226

RPM18226 cells were grown under the same conditions as the MOLT-4 cells.20,000 cells were seeded into 96 well plates for 24 hrs prior totreatment with increasing concentrations of compound 19 as described forMOLT-4 cells. As shown in FIG. 35D, the growth of the cells under 5% FBSand FBS fat free was similar except that 5 μM compound 19 in 5% FF FBSinhibited growth about 1.5 more than the corresponding amount in 5% FBS.Compound 19 was similarly effective at 10 μM and 20 μM in 5% FBS and 5%FF FBS. There was a strong trend of inhibition starting at 3 μM in 5%FBS condition (FIG. 35E) and FBS fat free serum at 2 μM (FIG. 35F).

Example 16 Compound 17 Inhibits the Growth of Human Acute LymphoblasticLeukemia Cell Line MOLT-4

MOLT-4 cells were grown at two different conditions under 5% fetalbovine serum (FBS) and under 5% FBS fat free serum (charcoal treatedserum). 20,000 cells were seeded into a 96 well plate for 24 hoursbefore starting the treatment with different concentrations of compound17. Compound 17 dissolved in DMSO was added to cells for 48 hours beforedetermining cell growth using MTT assay. As shown in FIG. 36A, thegrowth of the cells under 5% FBS and 5% FBS fat free was stronglyinhibited at 5, 10 and 20 μM. Under 5% FF FBS, 3 μM exhibited about 2.5times the inhibition as under 5% FBS conditions. Compound 17 inhibitedcell growth about 85-100% at 5, 10 and 20 μM under both growthconditions. Under both 5% FBS and 5% FBS fat free sera there was astrong trend of inhibition starting at 1 μM (FIGS. 36B-36C).

Compound 17 Inhibits the Growth of Human Multiple Myeloma Cell Line RPMI

RPM18226 cells were grown under the same conditions as the MOLT-4 cells.20,000 cells were seeded into 96 well plates for 24 hrs prior totreatment with increasing concentrations of compound 19 as described forMOLT-4 cells. As shown in FIG. 36D, the growth of the cells under 5% FBSand FBS fat free was initially similar except that compound 17 in 5% FFFBS inhibited growth about 1.5 to 6 times more than the correspondingamount in 5% FBS at 5, 10 and 20 μM. There was a strong trend ofinhibition starting at 3 μM in 5% FBS condition (FIG. 36E) and 5% FBSfat free serum (FIG. 36F).

The following references are cited herein.

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Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, composition of matter, means,methods and steps described in the specification. As one of ordinaryskill in the art will readily appreciate from the disclosure of thepresent invention, processes, compositions of matter, means, methods, orsteps, presently existing or later to be developed that performsubstantially the same function or achieve substantially the same resultas the corresponding embodiments described herein may be utilizedaccording to the present invention.

What is claimed is:
 1. A method for treating a disease related to cellhyper-proliferation in a patient in need thereof, comprising the step ofadministering to the patient, in a pharmaceutically acceptable medium, atherapeutically effective amount of a compound having a structure:A-B—C wherein A is a pyridine or a substituted pyridine, a piperidine ora substituted piperidine, a pyrrolidine or a substituted pyrrolidine, athiazole or a substituted thiazole, a phenyl ring or a substitutedphenyl ring; B is a thioazole or a substituted thioazole, a piperazineor a substituted piperazine, a phenyl ring or a substituted phenyl ring;and C is a phenyl ring or a substituted phenyl ring, a pyridine or asubstituted pyridine, a thioazole or a substituted thioazole; or apharmaceutically acceptable salt or a stereoisomer thereof or acombination thereof.
 2. The method of claim 1, wherein the chemicalstructure of the compound is:

wherein R₁ is H, halogen, —OH, C₁₋₃alkyl, —OR₃, —NHR₅ R₃ is C₁-C₃ alkyl,Bz or —OCH₂—C(O)OR₄; R₄ is H or C₁-C₃ alkyl; R₅ is H, C₁-C₄ alkyl,methylcyclopropane, cyclohexane, benzyl, —NHC(O)R₆, or —SO₂—R₇; R₆ isC₁-C₃alkyl or —O-tert-butyl; R₇ is methyl, thiophenyl or quinolinyl; R₂is C₂-C₄alkyl or —C(O)OR₈, and R₈ is C₃-C₅ alkyl or aryl; or apharmaceutically acceptable salt or a stereoisomer thereof or acombination thereof.
 3. The method of claim 2, wherein said halogen isbromine.
 4. The method of claim 2, wherein the compound is:4-(4-bromophenyl)-2-(pyrrolidin-2-yl)thiazole,4-(4-bromophenyl)-2-(1-propylpyrrolidin-2-yl)thiazole, tert-butyl2-(4-(4-bromophenyl)thiazol-2-yl)pyrrolidine-1-carboxylate, benzyl2-(4-(4-bromophenyl)thiazol-2-yl)pyrrolidine-1-carboxylate, orbenzyl(R)-2-(4-(4-(methylsulfonamido)phenyl)thiazol-2-yl)pyrrolidine-1-carboxylate.5. The method of claim 1, wherein the chemical structure of the compoundis:

wherein R₁ is H, halogen, —OH, C₁₋₃alkyl, —OR₃, or —NHR₅; R₃ is C₁-C₃alkyl, Bz or —OCH₂—C(O)OR₄; R₄ is H or C₁-C₃ alkyl; R₅ is H, C₁-C₄alkyl, methylcyclopropane, cyclohexane, benzyl, —C(O)R₆, or —SO₂—R₇; R₆is C₁-C₃alkyl or —O-tert-butyl; R₇ is methyl, thiophenyl or quinolinyl;R₂ is C₂-C₄alkyl or —C(O)OR₈, and R₈ is C₃-C₅ alkyl or aryl; or apharmaceutically acceptable salt or a stereoisomer thereof or acombination thereof.
 6. The method of claim 5, wherein said halogen isbromine.
 7. The method of claim 5, wherein the compound is:3-(4-(4-bromophenyl)thiazol-2-yl)-1-propylpiperidine, tert-butyl3-(4-(4-bromophenyl)thiazol-2-yl)piperidine-1-carboxylate, benzyl3-(4-(4-bromophenyl)thiazol-2-yl)piperidine-1-carboxylate, benzyl4-(4-(4-bromophenyl)thiazol-2-yl)piperidine-1-carboxylate, benzyl3-(4-(4-(methylsulfonamido)phenyl)thiazol-2-yl)piperidine-1-carboxylate,or benzyl4-(4-(4-(methylsulfonamido)phenyl)thiazol-2-yl)piperidine-1-carboxylate.8. The method of claim 1, wherein the chemical structure of the compoundis:

wherein R₉ is H, halogen, —OH, —OR₁₁ or —NHR₁₃; R₁₁ is C₁-C₃ alkyl, Bzor —OCH₂—C(O)OR₁₂; R₁₂ is H or C₁-C₃ alkyl; R₁₃ is H, C₁-C₄ alkyl,methylcyclopropane, cyclohexane, benzyl, or —C(O)R₁₄, or —SO₂—R₁₅; R₁₄is C₁-C₃alkyl or —O-tert-butyl; R₁₅ is methyl, thiophenyl, quinolinyl,or phenylR₁₆; R₁₆ is 4-chloro, 4-methyl or 3-chloro-4-methyl; R₁₀ isnitrogen or methylene, n is 0 or 1 and when n is 1, Z is —C═O; and A is

wherein R₁₇ is H or C₁-C₃ alkyl group; or a pharmaceutically acceptablesalt or a stereoisomer thereof or a combination thereof.
 9. The methodof claim 8, wherein the compound is:3-chloro-4-methyl-N-(6-(4-(3-(trifluoromethyl)benzyl)piperazin-1-yl)pyridin-3-yl)benzenesulfonamide,(4-(5-chloro-2-methylphenyl)piperazin-1-yl)(4-(tosylamino)phenyl)methanone,4-(4-((1-methyl-1H-benzo[d]imidazole-2-yl)methyl)piperazin-1-yl)-N-tosylbenzenamine,or4-chloro-N-(4-(4-((1-methyl-1H-benzo[d]imidazol-2-yl)methyl)piperazin-1-yl)phenyl)benzenesulfonamide.10. The method of claim 1, wherein the chemical structure of thecompound is:

The method of claim 10, wherein the compound is:4-(3-(pyridin-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)-N-tosylbenzenamine;(Z)-4-(3-cyano-3-(4-(2,4-dimethylphenyl)thiazol-2-yl)allyl)-N-(thiazol-2-yl)benzenesulfonamide;N-(3-(H-imidazo[1,2-a]pyridine-2-yl)phenyl)-4-methyl-2-phenylthiazole-5-carboxamide;N-(3-(benzo[d]thiazol-2-yl)phenyl)isonicotinamide;3-(4-chlorophenyl)-4,5-dihydro-1-phenyl-5-(2-phenylthiazol-4-yl)-1H-pyrazole;N-(4-(6-methylbenzo[d]thiazol-2-yl)phenyl)-2-(N-m-tolylmethylsulfonamido)acetamide;orN-(4-(6-methylbenzo[d]thiazol-2-yl)phenyl)-2-(N-p-tolylmethylsulfonamido)acetamide.11. The method of claim 1, wherein the disease related to cellhyper-proliferation is cancer.
 12. The method of claim 11, wherein thecancer is a cancer of the breast, respiratory tract, brain, reproductiveorgans, prostate, digestive tract, urinary tract, eye, liver, skin, headand neck, thyroid, parathyroid, lymphoma, sarcoma, melanoma, leukemia ora distant metastasis of a solid tumor.
 13. A method for treating acancer in a patient in need thereof, comprising the step ofadministering to the patient, in a pharmaceutically acceptable medium, atherapeutically effective amount of a compound having the chemicalstructure:

wherein R is H, isopropyl, benzyl, cyclohexyl, cyclopropylmethyl, —COMe,tert-butyloxycarbonyl, or methylsulfonyl, or a pharmaceuticallyacceptable salt or a stereoisomer thereof or a combination thereof. 14.The method of claim 13, wherein the chemical structure of the compoundis:


15. The method of claim 13, wherein the compound is:4-(4-bromophenyl)-2-(1-propylpyrrolidin-2-yl)thiazole,N-(4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenyl)methanesulfonamide,4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)benzenamine,N-isopropyl-4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)benzenamine,N-(4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenyl)acetamide,N-(cyclopropylmethyl)-4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)benzenamine,N-benzyl-4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)benzenamine,N-cyclohexyl-4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)benzenamine,4-(3-(pyridin-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)-N-tosylbenzenamine,(Z)-4-(3-cyano-3-(4-(2,4-dimethylphenyl)thiazol-2-yl)allyl)-N-(thiazol-2-yl)benzenesulfonamide,N-(4-(6-methylbenzo[d]thiazol-2-yl)phenyl)-2-(N-m-tolylmethylsulfonamido)acetamide,orN-(4-(6-methylbenzo[d]thiazol-2-yl)phenyl)-2-(N-p-tolylmethylsulfonamido)acetamide.16. The method of claim 13, wherein the cancer is a cancer of therespiratory tract, brain, reproductive organs, digestive tract, urinarytract, eye, liver, skin, head and neck, thyroid, parathyroid, lymphoma,sarcoma, melanoma, leukemia or a distant metastasis of a solid tumor.17. A method for treating a disease related to cell hyper-proliferationin a patient in need thereof, comprising the step of administering tothe patient, in a pharmaceutically acceptable medium, a therapeuticallyeffective amount of a compound that is:4-(4-bromophenyl)-2-(pyrrolidin-2-yl)thiazole,4-(4-bromophenyl)-2-(1-propylpyrrolidin-2-yl)thiazole, tert-butyl2-(4-(4-bromophenyl)thiazol-2-yl)pyrrolidine-1-carboxylate, benzyl2-(4-(4-bromophenyl)thiazol-2-yl)pyrrolidine-1-carboxylate,benzyl(R)-2-(4-(4-(methylsulfonamido)phenyl)thiazol-2-yl)pyrrolidine-1-carboxylate,3-(4-(4-bromophenyl)thiazol-2-yl)-1-propylpiperidine, tert-butyl3-(4-(4-bromophenyl)thiazol-2-yl)piperidine-1-carboxylate, benzyl3-(4-(4-bromophenyl)thiazol-2-yl)piperidine-1-carboxylate, benzyl4-(4-(4-bromophenyl)thiazol-2-yl)piperidine-1-carboxylate, benzyl3-(4-(4-(methylsulfonamido)phenyl)thiazol-2-yl)piperidine-1-carboxylate,benzyl4-(4-(4-(methylsulfonamido)phenyl)thiazol-2-yl)piperidine-1-carboxylate,3-chloro-4-methyl-N-(6-(4-(3-(trifluoromethyl)benzyl)piperazin-1-yl)pyridin-3-yl)benzenesulfonamide,(4-(5-chloro-2-methylphenyl)piperazin-1-yl)(4-(tosylamino)phenyl)methanone,4-(4-((1-methyl-1H-benzo[d]imidazole-2-yl)methyl)piperazin-1-yl)-N-tosylbenzenamine,4-chloro-N-(4-(4-((1-methyl-1H-benzo[d]imidazol-2-yl)methyl)piperazin-1-yl)phenyl)benzenesulfonamide,4-(3-(pyridin-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)-N-tosylbenzenamine;(Z)-4-(3-cyano-3-(4-(2,4-dimethylphenyl)thiazol-2-yl)allyl)-N-(thiazol-2-yl)benzenesulfonamide;N-(3-(H-imidazo[1,2-a]pyridine-2-yl)phenyl)-4-methyl-2-phenylthiazole-5-carboxamide;N-(3-(benzo[d]thiazol-2-yl)phenyl)isonicotinamide;3-(4-chlorophenyl)-4,5-dihydro-1-phenyl-5-(2-phenylthiazol-4-yl)-1H-pyrazole;N-(4-(6-methylbenzo[d]thiazol-2-yl)phenyl)-2-(N-m-tolylmethylsulfonamido)acetamide;orN-(4-(6-methylbenzo[d]thiazol-2-yl)phenyl)-2-(N-p-tolylmethylsulfonamido)acetamide.18. The method of claim 17, wherein the disease related to cellhyper-proliferation is cancer.
 19. The method of claim 18, wherein thecancer is a cancer of the breast, respiratory tract, brain, reproductiveorgans, prostate, digestive tract, urinary tract, eye, liver, skin, headand neck, thyroid, parathyroid, lymphoma, sarcoma, melanoma, leukemia ora distant metastasis of a solid tumor.